Immunosuppressive polypeptides and nucleic acids

ABSTRACT

The invention provides immunosuppressive polypeptides and nucleic acids encoding such polypeptides. In one aspect, the invention provides mutant CTLA-4 polypeptides and nucleic acids encoding mutant CTLA-4 polypeptides. Compositions and methods for utilizing such polypeptides and nucleic acids are also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalPatent Application Ser. No. 60/984,631, filed on Nov. 1, 2007, and U.S.Provisional Patent Application Ser. No. 61/051,215, filed on May 7,2008, the disclosure of each of which is incorporated herein byreference in its entirety for all purposes.

COPYRIGHT NOTIFICATION

Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of thisdisclosure contains material which is subject to copyright protection.The copyright owner has no objection to the facsimile reproduction byanyone of the patent document or patent disclosure, as it appears in thePatent and Trademark Office patent file or records, but otherwisereserves all copyright rights whatsoever.

FIELD OF THE INVENTION

This invention relates generally to novel polypeptides that bind CD80and/or CD86, nucleic acid encoding such polypeptides, and methods ofmaking and using such polypeptides and nucleic acids.

BACKGROUND OF THE INVENTION

T cells play a major role in the initiation and regulation of immuneresponses. For complete activation of T cells to occur, at least twodistinct signaling events are required. A first signal is produced bythe interaction of T cell receptors (TCR) expressed on T cells withspecific antigens (Ag) presented in the context of majorhistocompatibility complex (MHC) molecules expressed onantigen-presenting cells (APCs). A second (co-stimulation) signalresults from the interaction between co-stimulatory ligands expressed onAPCs and their corresponding receptors expressed on T cells. A dominantco-stimulation pathway involves the interaction between the CD80 (B7-1or B7.1) and CD86 (B7-2 or B7.2) ligands expressed on APCs with CD28 andCTLA-4 (also known as CD152) expressed mainly on T cells. CTLA-4(cytotoxic T-lymphocyte antigen 4) and CD28 serve as receptors for theCD80 and CD86 ligands.

Positive signaling is mediated through the CD28 receptor. Binding of theCD80 and/or CD86 ligand(s) to CD28 lowers the threshold of T cellactivation by promoting the formation of immunological synapses (ViolaA. et al., Science 283:680-682 (1999)). Additionally, CD28co-stimulation activates or enhances the production of factors centralto T cell proliferation and survival, such as interleukin-2 (IL-2),NF-κB, and Bcl-XL (Norton S. D. et at., J. Immunol. 149:1556-1561(1992); Vella A. T. et al., J. Immunol. 158: 4714-4720 (1997)). In vivo,CD28-deficient mice are severely immunocompromised and show poorantigen-specific T cell responses (Green, J. M. et al., Immunity1:501-508 (1994)). T cell anergy or tolerance may result when T cellsare activated in the absence of the costimulatory signal.

Negative signaling is mediated through the CTLA-4 receptor. The CD80 andCD86 ligands each bind with high avidity to CTLA-4 and counterbalanceimmunoproliferative responses derived from CD28 signaling. Potentialmechanisms of CTLA-4 signaling include competitive binding ofco-stimulatory molecules CD80/CD86 (Masteller, E. M. et al., J. Immunol.164:5319 (2000)), inhibition of TCR signaling by induction ofphosphatases to the immunosynapse (Lee K. M. et al., Science 282:2263(1998)), and disruption of the immunological synapse (Pentcheva-Hoang T.et al., Immunity 21:401 (2004); Chikuma S. et al., J. Exp. Med. 197:129(2003); Schneider H. et al., Science 313: 1972 (2006)). In vivo, CTLA-4deficient mice show profound autoimmune phenotypes characterized bymassive tissue infiltration and organ destruction (Waterhouse P. et al.,Science 270:985 (1995)).

Therapeutic agents designed to antagonize the CD80/CD86 co-stimulationpathway, such as soluble human CTLA-4-Ig, hold promise for the treatmentof autoimmune diseases and disorders. The present invention providesadvantageous molecules having improved abilities to modulate or suppresssignaling through the CD80/CD86 co-stimulation pathway and methods ofusing such molecules for selected and differential manipulation of Tcell responses. Such molecules are of beneficial use in a variety ofapplications as discussed in detail below.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides an isolated or recombinantCTLA-4 polypeptide comprising a polypeptide sequence which differs fromthe polypeptide sequence of the extracellular domain of human CTLA-4shown in SEQ ID NO:159 in up to 15 amino acid residues, wherein theisolated or recombinant CTLA-4 polypeptide has an ability to bind CD80or CD86 or an extracellular domain of either and/or has an ability tosuppress or inhibit an immune response.

A polypeptide according to the first aspect of the invention may have atleast 90% sequence identity, or at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence identity, to the polypeptidesequence of SEQ ID NO:36. A polypeptide according to the first aspect ofthe invention may comprise the polypeptide sequence of SEQ ID NO:36.

A polypeptide according to the first aspect of the invention may have atleast 90% sequence identity, or at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence identity, to the polypeptidesequence of SEQ ID NO:50. A polypeptide according to the first aspect ofthe invention may comprise the polypeptide sequence of SEQ ID NO:50.

A polypeptide according to the first aspect of the invention may have anability to bind human CD80 or human CD86 or an extracellular domain ofeither.

A polypeptide according to the first aspect of the invention maycomprise a polypeptide sequence that is 124 amino acid residues inlength.

A polypeptide according to the first aspect of the invention maycomprise one amino acid substitution at an amino acid position selectedfrom the group consisting of amino acid positions corresponding toposition 50, 54, 55, 56, 64, 65, 70, or 85 relative to SEQ ID NO:159.

A polypeptide according to the first aspect of the invention maycomprise two, three, or four amino acid substitutions at amino acidpositions selected from the group consisting of amino acid positionscorresponding to position 50, 54, 55, 56, 64, 65, 70, or 85 relative toSEQ ID NO:159.

A polypeptide of the first aspect of the invention may comprise an aminoacid substitution at position 70 relative to SEQ ID NO:159, such as thesubstitution S70F. A polypeptide of the first aspect of the inventionmay comprise an amino acid substitution at position 104 relative to SEQID NO:159, such as the substitution L104E.

A polypeptide of the first aspect of the invention may comprise an aminoacid substitution at position 30 relative to SEQ ID NO:159, such as thesubstitution T30N/D/A or the substitution T30N.

A polypeptide of the first aspect of the invention may comprise an aminoacid substitution at position 64 relative to SEQ ID NO:159, such as thesubstitution S64P.

A polypeptide of the first aspect of the invention may comprise an aminoacid substitution at position 50 relative to SEQ ID NO:159, such as thesubstitution A50M.

A polypeptide of the first aspect of the invention may comprise an aminoacid substitution at position 54 relative to SEQ ID NO:159, such as thesubstitution M54K/V or the substitution M54K.

A polypeptide of the first aspect of the invention may comprise an aminoacid substitution at position 65 relative to SEQ ID NO:159, such as thesubstitution I65S.

A polypeptide of the first aspect of the invention may comprise an aminoacid substitution at position 56 relative to SEQ ID NO:159, such as thesubstitution N56D.

A polypeptide of the first aspect of the invention may comprise an aminoacid substitution at position 55 relative to SEQ ID NO:159, such as thesubstitution G55E.

A polypeptide of the first aspect of the invention may comprise an aminoacid substitution at positions 85 relative to SEQ ID NO:159, such as thesubstitution M85A.

A polypeptide of the first aspect of the invention may comprise an aminoacid substitution at position 24 relative to SEQ ID NO:159, such as thesubstitution A24E/S or the substitution A24E.

A polypeptide of the first aspect of the invention may have a bindingaffinity for CD86 or an extracellular domain thereof that is about equalto or greater than the binding affinity of a monomeric human CTLA-4extracellular domain for CD86 or CD86 extracellular domain.

A polypeptide of the first aspect of the invention may have a bindingaffinity for CD80 or an extracellular domain thereof that is greaterthan the binding affinity of a monomeric human CTLA-4 extracellulardomain for CD80 or CD80 extracellular domain.

A polypeptide of the first aspect of the invention may have an abilityto suppress an immune response.

A polypeptide of the first aspect of the invention may have an abilityto inhibit T cell activation or T cell proliferation.

In a second aspect, the invention provides an isolated or recombinantpolypeptide multimer comprising at least two polypeptides of the firstaspect of the invention.

In a third aspect, the invention provides an isolated or recombinantfusion protein comprising (a) a polypeptide according to the firstaspect of the invention, and (b) a second polypeptide, wherein thesecond polypeptide is an Ig Fc polypeptide, and wherein the fusionprotein has an ability to bind CD80 and/or CD86 or an extracellulardomain of either or both, and/or an ability to modulate or regulate animmune response.

In a fourth aspect, the invention provides an isolated or recombinantdimeric fusion protein comprising two monomeric fusion proteinsaccording to the third aspect of the invention.

In a fifth aspect, the invention provides an isolated or recombinantnucleic acid comprising a nucleotide sequence that encodes a polypeptideof the first aspect of the invention, a multimer of the second aspect ofthe invention, a fusion protein of the third aspect of the invention, ora dimeric fusion protein of the fourth aspect of the invention.

In a sixth aspect, the invention provides a vector comprising a nucleicacid of the fifth aspect of the invention.

In a seventh aspect, the invention provides an isolated or recombinanthost cell comprising a polypeptide of the first aspect of the invention,a multimer of the second aspect of the invention, a fusion protein ofthe third aspect of the invention, a dimeric fusion protein of thefourth aspect of the invention, a nucleic acid of the fifth aspect ofthe invention, and/or a vector of the sixth aspect of the invention.

In an eighth aspect, the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable excipient, pharmaceuticallyacceptable carrier, or pharmaceutically acceptable diluent and one ormore of the following: a polypeptide of the first aspect of theinvention, a multimer of the second aspect of the invention, a fusionprotein of the third aspect of the invention, a dimeric fusion proteinof the fourth aspect of the invention, a nucleic acid of the fifthaspect of the invention, a vector of the sixth aspect of the invention,and/or a host cell of the seventh aspect of the invention.

In a ninth aspect, the invention provides a method for suppressing animmune response, said method comprising contacting a B7-positive cellwith an effective amount of at least one of: a polypeptide of the firstaspect of the invention, a multimer of the second aspect of theinvention, a fusion protein of the third aspect of the invention, adimeric fusion protein of the fourth aspect of the invention, a nucleicacid of the fifth aspect of the invention, a vector of the sixth aspectof the invention, and/or a host cell of the seventh aspect of theinvention, to suppress an immune response, wherein an immune response isthereby suppressed.

In a tenth aspect, the invention provides a polypeptide of the firstaspect of the invention, a multimer of the second aspect of theinvention, a fusion protein of the third aspect of the invention, adimeric fusion protein of the fourth aspect of the invention, a nucleicacid of the fifth aspect of the invention, a vector of the sixth aspectof the invention, and/or a host cell of the seventh aspect of theinvention, for use in suppressing an immune response.

In an eleventh aspect, the invention provides the use of a polypeptideof the first aspect of the invention, a multimer of the second aspect ofthe invention, a fusion protein of the third aspect of the invention, adimeric fusion protein of the fourth aspect of the invention, a nucleicacid of the fifth aspect of the invention, a vector of the sixth aspectof the invention, and/or a host cell of the seventh aspect of theinvention, in the manufacture of a medicament for suppressing an immuneresponse.

In a twelfth aspect, the invention provides a conjugate comprising apolypeptide of the first aspect of the invention, a multimer of thesecond aspect of the invention, a fusion protein of the third aspect ofthe invention, or a dimeric fusion protein of the fourth aspect of theinvention, and a non-polypeptide moiety covalently attached to suchpolypeptide, multimer, fusion protein, or dimeric fusion protein,wherein said conjugate has an ability to suppress an immune response.

Other aspects of the invention are described below.

In another aspect, the invention provides an isolated or recombinantpolypeptide comprising a polypeptide sequence having at least 95%, 96%,97%, 98%, 99%, or 100% sequence identity to at least one polypeptidesequence selected from the group consisting of SEQ ID NOS:1-73, whereinthe polypeptide binds CD80 or CD86 or an extracellular domain (ECD) ofeither, and/or has an ability to suppress or inhibit an immune response.

In another aspect, the invention provides an isolated or recombinantpolypeptide comprising a polypeptide sequence having at least 95%, 96%,97%, 98%, 99%, or 100% sequence identity to at least one polypeptidesequence selected from the group consisting of SEQ ID NOS:1-73, whereinthe polypeptide has a binding affinity for a human CD86 extracellulardomain or human CD80 extracellular domain that is about equal to orgreater than the binding affinity of a human CTLA-4 extracellular domainfor the human CD86 extracellular domain or human CD80 extracellulardomain, respectively, and wherein the polypeptide optionally has anability to suppress an immune response. Some such polypeptides have abinding affinity for the human CD86 extracellular domain that is greaterthan the binding affinity of the human CTLA-4 extracellular domain forthe human CD86 extracellular domain. Some such polypeptides have abinding affinity for the human CD80 extracellular domain that is greaterthan the binding affinity of the human CTLA-4 extracellular domain forthe human CD80 extracellular domain.

In another aspect, the invention provides an isolated or recombinantmutant CTLA-4 polypeptide comprising a polypeptide sequence which (a)differs from the polypeptide sequence of the extracellular domain ofhuman CTLA-4 shown in SEQ ID NO:159 in no more than 10, 9, 8, 7, or 6amino acid residues (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acid residues), and (b) comprises at least one amino acidsubstitution at an amino acid position corresponding to position 50, 54,55, 56, 64, 65, 70, or 85 relative to SEQ ID NO:159, wherein the mutantCTLA-4 polypeptide has an ability to bind CD80 or CD86 or anextracellular domain of either, and/or has an ability to suppress orinhibit an immune response.

In another aspect, the invention provides an isolated or recombinantpolypeptide which comprises a polypeptide sequence comprising (i) atleast 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to apolypeptide sequence selected from the group consisting of SEQ IDNOS:1-73 and (ii) a phenylalanine residue at an amino acid positioncorresponding to position 70 of said polypeptide sequence selected fromthe group consisting of SEQ ID NO:1-73, wherein the polypeptide has anability to bind CD80 and/or CD86 or an extracellular domain of either orboth, and/or has an ability to suppress or inhibit an immune response.

In another aspect, the invention provides an isolated or recombinantmutant CTLA-4 polypeptide that binds CD80 and/or CD86 and/or anextracellular domain of either or both, and/or is capable of suppressingan immune response, wherein said polypeptide comprises a polypeptidesequence which (a) differs from the polypeptide sequence of human CTLA-4extracellular domain polypeptide shown in SEQ ID NO:159 in no more than10, 9, 8, 7, or 6 amino acid residues (e.g., no more than 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 amino acid residues), and (b) comprises at least oneamino acid substitution, wherein said at least amino acid substitutioncomprises S70F, wherein amino acid residue positions are numberedaccording to SEQ ID NO:159.

In another aspect, the invention provides an isolated or recombinantmutant CTLA-4 polypeptide comprising a polypeptide sequence which (a)differs from the polypeptide sequence of the extracellular domain ofhuman CTLA-4 shown in SEQ ID NO:159 in no more than 11, 10, 9, 8, 7, or6 amino acid residues (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11 amino acid residues), and (b) comprises at least one amino acidsubstitution at an amino acid residue position corresponding to position24, 30, 32, 50, 54, 55, 56, 64, 65, 70, or 85 relative to SEQ ID NO:159,wherein the mutant CTLA-4 polypeptide has an ability to bind CD80 orCD86 or an extracellular domain of either, and/or has an ability tosuppress or inhibit an immune response.

In another aspect, the invention provides an isolated or recombinantpolypeptide comprising a polypeptide sequence which (a) differs from thepolypeptide sequence shown in SEQ ID NO:31 in no more than 10, 9, 8, 7,or 6 amino acid residues (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 amino acid residues), and (b) comprises at least one of thefollowing: a methionine residue at a position corresponding to position50 of SEQ ID NO:31, a lysine residue at a position corresponding toposition 54 of SEQ ID NO:31, a glutamic acid residue at a positioncorresponding to position 55 of SEQ ID NO:31, a proline residue at aposition corresponding to position 64 of SEQ ID NO:31, a serine residueat a position corresponding to position 65 of SEQ ID NO:31, aphenylalanine residue at a position corresponding to position 70 of SEQID NO:31, wherein amino acid residue positions are numbered according toSEQ ID NO:31, and the polypeptide binds CD80 and/or CD86 and/or an ECDof either or both, and/or inhibits an immune response.

In another aspect, the invention provides an isolated or recombinantfusion protein dimer comprising two monomeric fusion proteins linked viaat least one disulfide bond formed between two cysteine residues presentin each monomeric mutant fusion protein, wherein each monomeric fusionprotein comprises (a) a polypeptide comprising a polypeptide sequencehaving at least 95%, 96%, 97%, 98%, 99%, or 100% identity to at leastone polypeptide sequence selected from the group consisting of SEQ IDNOS:1-73 and (b) an Ig Fc polypeptide, wherein the fusion protein dimerhas an ability to bind CD80 and/or CD86, and/or CD80-Ig and/or CD86-Ig,and/or has an ability to inhibit or suppress an immune response.

In another aspect, the invention provides an isolated or recombinantfusion protein dimer comprising two monomeric fusion proteins, each suchmonomeric fusion protein comprising a polypeptide sequence having atleast 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least onepolypeptide sequence selected from the group consisting of SEQ IDNOS:74-79, 197-200, 205-214, and 219-222, wherein the polypeptide bindsCD80 and/or CD86 and/or an extracellular domain thereof and/orsuppresses an immune response, or a complementary polynucleotidesequence thereof.

In another aspect, the invention provides an isolated or recombinantfusion protein dimer comprising two monomeric fusion proteins, whereineach monomeric fusion protein comprises: (1) a polypeptide comprising apolypeptide sequence which differs from a polypeptide sequence selectedfrom the group consisting of SEQ ID NOS:1-73 in no more than 10, 9, 8,7, or 6 amino acid residues (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 amino acid residues), and wherein the amino acid residue in thepolypeptide sequence at position 41, 50, 54, 55, 56, 64, 65, 70, or 85is identical to the amino acid residue at the corresponding position ofsaid selected polypeptide sequence, and (2) an Ig Fc polypeptide,wherein the fusion protein dimer binds CD80 and/or CD86, and/or inhibitsan immune response.

In another aspect, the invention provides an isolated or recombinantfusion protein dimer comprising two monomeric fusion proteins, whereineach monomeric fusion protein comprises: (1) a mutant CTLA-4extracellular domain polypeptide comprising a polypeptide sequence which(i) differs from the polypeptide sequence of the extracellular domain ofhuman CTLA-4 shown in SEQ ID NO:159 in no more than 10, 9, 8, 7, or 6amino acid residues (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acid residues), and (ii) comprises at least one amino acidsubstitution at an amino acid position corresponding to position 50, 54,55, 56, 64, 65, 70, or 85 relative to the polypeptide sequence of SEQ IDNO:159; and (2) an Ig Fc polypeptide, wherein the fusion protein dimerbinds CD80 and/or CD86, and/or suppresses or inhibits an immuneresponse. Some such fusion protein dimers comprise one or moresubstitutions at amino acid positions relative to SEQ ID NO:159 selectedfrom the group consisting of A50M, M54K, G55E, N56D, S64P, I65S, andS70F; and (2) an Ig Fc polypeptide, which Ig Fc polypeptide optionallyis IgG2 Fc polypeptide, wherein the mutant CTLA-4-Ig dimer binds hCD80and/or hCD86, and/or suppresses or inhibits an immune response.

In another aspect, the invention provides an isolated or recombinantpolypeptide comprising a polypeptide sequence having at least 95%, 96%,97%, 98%, 99%, or 100% sequence identity to a polypeptide sequenceselected from the group consisting of SEQ ID NO:26, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:50, and SEQ ID NO:56, wherein the polypeptide (i) bindsCD80 and/or CD86 or an extracellular domain of either or both, and/or(ii) suppresses an immune response.

In another aspect, the invention provides an isolated or recombinantpolypeptide comprising a polypeptide sequence having at least 95%, 96%,97%, 98%, 99%, or 100% sequence identity to a polypeptide sequenceselected from the group consisting of SEQ ID NOS:74-79, 197-200,205-214, and 219-222, wherein the polypeptide (i) binds CD80 and/or CD86or an extracellular domain of either or both, (ii) binds a CD80-Igfusion protein and/or CD86-Ig fusion protein, and/or (iii) suppresses animmune response.

Also provided is an isolated or recombinant fusion protein dimercomprising two monomeric fusion proteins linked via at least onedisulfide bond formed between two cysteine residues present in eachmonomeric mutant fusion protein, wherein each monomeric fusion proteincomprises (a) a polypeptide comprising a polypeptide sequence having atleast 95% identity to at least one polypeptide sequence selected fromthe group consisting of SEQ ID NO:79, SEQ ID NO:197, SEQ ID NO:198, SEQID NO:199, and SEQ ID NO:200 and (b) an Ig Fc polypeptide, wherein thefusion protein dimer has (i) an ability to bind CD80 and/or CD86 and/oran extracellular domain of CD80 and/or CD86, (ii) an ability to bindCD80-Ig and/or CD86-Ig, and/or (iii) has an ability to inhibit orsuppress an immune response.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence that encodes apolypeptide comprising a polypeptide sequence having at least 95%, 96%,97%, 98%, 99% or 100% sequence identity to at least one polypeptidesequence selected from the group consisting of SEQ ID NOS:1-79, 197-200,205-214, and 219-222, wherein the polypeptide binds CD80 and/or CD86and/or an extracellular domain of either or both, and/or has an abilityto suppress an immune response, or a complementary polynucleotidesequence thereof.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence that encodes a fusionprotein comprising a polypeptide sequence having at least 95%, 96%, 97%,98%, 99% or 100% sequence identity to at least one polypeptide sequenceselected from the group consisting of SEQ ID NOS: 74-79, 197-200,205-214, and 219-222, wherein the polypeptide binds CD80 and/or CD86and/or an extracellular domain thereof and/or suppresses an immuneresponse, or a complementary polynucleotide sequence thereof.

In another aspect, the invention provides an isolated of recombinantnucleic acid comprising: (a) a polynucleotide sequence having at least95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least onepolynucleotide sequence selected from the group consisting of SEQ IDNOS:80-158, 201-204, 223, and 224; (b) a complementary polynucleotidesequence of (a); or (c) a fragment of any polynucleotide sequence of (a)or (b), wherein the nucleic acid encodes a polypeptide that binds CD80and/or CD86 and/or an extracellular domain of either or both, and/or hasan ability to suppress or inhibit an immune response.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes apolypeptide which comprises a polypeptide sequence (a) which differsfrom a polypeptide sequence selected from the group consisting of SEQ IDNOS:1-73 in no more than 10, 9, 8, 7, or 6 amino acid residues (e.g., nomore than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues), and (b)wherein the amino acid residue in the polypeptide sequence at position41, 50, 54, 55, 56, 64, 65, 70, or 85 is identical to the amino acidresidue at the corresponding position of said polypeptide sequenceselected from the group consisting of SEQ ID NOS:1-73, wherein thepolypeptide binds CD80 and/or CD86 and/or an extracellular domain ofeither or both, and/or inhibits an immune response, or a complementarypolynucleotide sequence thereof.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes apolypeptide comprising a polypeptide sequence which (a) differs from thepolypeptide sequence of the extracellular domain of human CTLA-4 shownin SEQ ID NO:159 in no more than 10, 9, 8, 7, or 6 amino acid residues(e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidresidues), and (b) comprises at least one amino acid substitution at anamino acid position corresponding to position 50, 54, 55, 56, 64, 65,70, or 85 relative to SEQ ID NO:159, wherein said polypeptide has anability to bind CD80 and/or CD86 and/or an extracellular domain ofeither, and/or has an ability to suppress or inhibit an immune response,or a complementary polynucleotide sequence thereof.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes apolypeptide comprising a polypeptide sequence having (i) at least 95%,96%, 97%, 98%, 99%, or 100% identity to a polypeptide sequence selectedfrom the group consisting of SEQ ID NOS:1-73 and (ii) a phenylalanineresidue at an amino acid position corresponding to position 70 of saidpolypeptide sequence selected from the group consisting of SEQ IDNO:1-73, wherein the polypeptide binds hCD80 and/or hCD86 or an ECDthereof and/or inhibits an immune response, wherein said amino acidsubstitution optionally comprises S70F, or a complementarypolynucleotide sequence thereof.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes arecombinant polypeptide dimer comprising two polypeptides, wherein eachsuch polypeptide comprises a polypeptide sequence having at least 95%,96%, 97%, 98%, 99%, or 100% identity to a sequence selected from thegroup consisting of SEQ ID NOS:1-73, wherein the dimer binds hCD80and/or hCD86 and/or inhibits an immune response, or a complementarypolynucleotide sequence thereof.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes a fusionprotein comprising (a) a polypeptide comprising a polypeptide sequencethat has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to at leastone polypeptide sequence selected from the group consisting of SEQ IDNOS:1-73, and (b) an Ig polypeptide, wherein the fusion protein bindsCD80 and/or CD86, and/or has an ability to suppress an immune response,or a complementary polynucleotide sequence thereof.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes apolypeptide comprising a polypeptide sequence which (a) differs from thepolypeptide sequence shown in SEQ ID NO:31 in no more than 10, 9, 8, 7,or 6 amino acid residues (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 amino acid residues), and (b) comprises at least one of thefollowing: a methionine residue at a position corresponding to position50 of SEQ ID NO:31, a lysine residue at a position corresponding toposition 54 of SEQ ID NO:31, a glutamic acid residue at a positioncorresponding to position 55 of SEQ ID NO:31, a proline residue at aposition corresponding to position 64 of SEQ ID NO:31, a serine residueat a position corresponding to position 65 of SEQ ID NO:31, aphenylalanine residue at a position corresponding to position 70 of SEQID NO:31, wherein amino acid residue positions are numbered according toSEQ ID NO:31, and wherein the polypeptide binds CD80 and/or CD86, and/orinhibits an immune response, or a complementary polynucleotide sequencethereof.

In another aspect, the invention provides an expression vectorcomprising: (i) a first polynucleotide sequence that encodes a firstpolypeptide comprising a polypeptide sequence having at least 95%, 96%,97%, 98%, 99%, or 100% identity to at least one polypeptide sequenceselected from the group consisting of SEQ ID NOS:1-73, wherein saidfirst polypeptide binds human CD86 and/or human CD80 and/or anextracellular domain of either or both, and/or suppresses an immuneresponse, and (ii) a second polynucleotide sequence that encodes asecond polypeptide comprising a hinge region, a CH2 domain, and a CH3domain of an immunoglobulin (Ig) polypeptide, which Ig polypeptide isoptionally human IgG2 Fc polypeptide.

In another aspect, the invention provides an isolated or recombinanthost cell transfected with a nucleic acid encoding a fusion protein, thenucleic acid comprising: (i) a first nucleotide sequence encoding afirst polypeptide comprising a polypeptide sequence having at least 95%,96%, 97%, 98%, 99%, or 100% identity to at least one polypeptidesequence selected from the group consisting of SEQ ID NOS:1-73, whereinsaid first polypeptide has an ability to bind human CD86 and/or humanCD80 and/or an extracellular domain of either or both, and/or has anability to suppress an immune response; and (ii) a second nucleotidesequence encoding a second polypeptide comprising a hinge region, a CH2domain, and a CH3 domain of an immunoglobulin (Ig) polypeptide, which Igpolypeptide is optionally human IgG2 Fc polypeptide, wherein the hostcell is capable of expressing the fusion protein.

In another aspect, the invention provides a method of suppressing animmune response, said method comprising contacting a B7-positive cellwith an effective amount of at least one polypeptide, conjugate, nucleicacid, vector, or cell of the invention to suppress an immune response,wherein an immune response is thereby suppressed.

In another aspect, the invention provides a method of modulating theinteraction of T cells expressing CD28 and/or CTLA-4 with B7-positivecells, the method comprising contacting B7-positive cells with aneffective amount of at least one polypeptide, conjugate, nucleic acid,vector, or cell of the invention to modulate the interaction ofB7-positive cells with CD28-positive T cells and/or CTLA-4-positive Tcells, wherein the interaction of B7-positive cells with CD28-positive Tcells and/or CTLA-4-positive T cells is modulated.

In another aspect, the invention provides a method of inhibiting theinteraction of CD28-positive T cells and/or CTLA-4-positive T cells withB7-positive cells, the method comprising contacting B7-positive cellswith an effective amount of at least one polypeptide, conjugate, nucleicacid, vector, or cell of the invention, wherein the interaction ofCD28-positive T cells and/or CTLA-4-positive T cells with B7-positivecells is inhibited.

In another aspect, the invention provides a method of inhibiting theinteraction of CD28-positive T cells with B7-positive cells in asubject, said method comprising administering to a subject an effectiveamount of at least one polypeptide, conjugate, nucleic acid, vector, orcell of the invention, wherein the interaction of endogenousCD28-positive T cells with endogenous B7-positive cells in the subjectis inhibited.

In another aspect, the invention provides a method of treating a subjecthaving an immune system disease or disorder modulated by interaction ofendogenous T cells with endogenous cells expressing CD80 and/or CD86,said method comprising administering to a subject in need of suchtreatment a therapeutically effective amount of at least onepolypeptide, conjugate, nucleic acid, vector, or cell of the invention,wherein interaction(s) between endogenous T cells and endogenous cellsexpressing said CD80 and/or said CD86 is inhibited, thereby treating theimmune system disease or disorder in the subject.

In another aspect, the invention provides a method of inhibitingrejection of a tissue or organ transplant from a donor by a recipientsubject, the method comprising administering to the recipient subject inneed thereof a therapeutically effective amount of at least onepolypeptide, conjugate, nucleic acid, vector, or cell of the invention,thereby inhibiting rejection of the tissue or organ transplant by therecipient subject.

In another aspect, the invention provides a method of making a fusionprotein, the method comprising: (1) culturing a host cell transformedwith a nucleic acid in a culture medium, wherein the nucleic acidcomprises (i) a first nucleotide sequence that encodes a polypeptidehaving at least 95%, 96%, 97%, 98%, 99%, or 100% identity to apolypeptide sequence of any of SEQ ID NOS:1-73, which polypeptide bindsCD86 and/or CD80, and/or an extracellular domain of either CD86 or CD80,and (ii) a second nucleotide sequence encoding an Ig polypeptidecomprising a hinge region, CH2 domain, and CH3 domain, whereby thenucleic acid is expressed and a fusion protein is produced; and (2)recovering the fusion protein.

Also provided is a method of producing a polypeptide comprisingintroducing into a population of cells a nucleic acid of the invention,wherein the nucleic acid is operatively linked to a regulatory sequenceeffective to produce the polypeptide encoded by the nucleic acid;culturing the cells in a culture medium to produce the polypeptide; andisolating the polypeptide from the cells or culture medium.

Also provided are compositions which comprise a molecule of theinvention (e.g., mutant CTLA-4 molecule) and an excipient, carrier, ordiluent. Also included are pharmaceutical compositions comprising amolecule of the invention and a pharmaceutically acceptable and anexcipient, carrier, or diluent.

Additional aspects of the invention are described below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of the plasmid expression vector pcDNAmutant CTLA-4-Ig, which comprises a nucleotide sequence encoding amutant CTLA-4-Ig fusion protein. In FIG. 1, each mutant CTLA-4-Ig fusionprotein comprises a mutant CTLA-4 ECD polypeptide of the invention fusedat its C-terminus to the N-terminus of a human IgG2 (hIgG2) Fcpolypeptide.

FIGS. 2A-2D are schematic diagrams of exemplary hCD80-Ig, hCD86-Ig,LEA29Y-Ig, and hCTLA-4-IgG2 fusion proteins, respectively. The signalpeptide, extracellular domain (ECD), linker (if any), and Ig Fc domainof each fusion protein are shown schematically. The amino acid residuespresent at the junctions between the signal peptide, ECD, linker (ifany), and Ig Fc are also shown. The signal peptide of each fusionprotein is typically cleaved during processing and thus the secreted(mature) fusion protein typically does not contain the signal peptidesequence. FIG. 2D presents a schematic diagram of a human CTLA-4-IgG2(“hCTLA-4-IgG2”) fusion protein comprising a human CTLA-4 extracellulardomain (“hCTLA-4 ECD”) covalently fused at its C-terminus to theN-terminus of a human IgG2 polypeptide. The predicted polypeptidesequence of this hCTLA-4-IgG2 fusion protein is shown in SEQ ID NO:161and comprises the following segments: hCTLA-4 signal peptide (amino acidresidues 1-37), hCTLA-4 ECD polypeptide (amino acid residues 38-161),and human IgG2 Fc polypeptide (amino acid residues 162-389). No linker(e.g., no amino acid residue(s)) is included between the C-terminus ofthe hCTLA-4 ECD polypeptide and the N-terminus of the human IgG2 Fc. Thehuman IgG2 Fc polypeptide comprises a hinge, CH2 domain, and CH3 domainof human IgG2. In FIG. 2D, the amino acid residues at the junctionsbetween these various segments are shown. Specifically, the last fouramino acid residues of the signal peptide, the first five and last fiveamino acid residues of the hCTLA-4 ECD polypeptide, and the first fiveand last five amino acid residues of the human IgG2 Fc polypeptide areshown.

The signal peptide is typically cleaved during processing and thus thesecreted fusion protein (mature fusion protein) of hCTLA-4-IgG2 does nottypically contain the signal peptide sequence. The polypeptide sequenceof the mature or secreted form of this hCTLA-4-IgG2 fusion protein isshown in SEQ ID NO:162. The sequence of the hCTLA-4 ECD polypeptidecomprises amino acid residues 1-124 of SEQ ID NO:162, and the sequenceof human IgG2 Fc polypeptide comprises amino acid residues 125-352 ofSEQ ID NO:162. In another aspect, this mature hCTLA-4 Ig fusion proteindoes not include the C-terminal lysine (K) residue and thus comprisesamino acid residues 1-351 of SEQ ID NO:162.

The mature hCTLA-4-IgG2 fusion protein, which has a total of 352 aminoacids, comprises amino acid residues 38-389 of the polypeptide sequenceof the full-length WT hCTLA-4 protein shown in SEQ ID NO:160, and beginswith the amino acid sequence: methionine-histidine-valine-alanine. Ifdesired, the amino acids of the mature form can be numbered beginningwith the Met of the Met-His-Val-Ala sequence, designating Met as thefirst residue (e.g., the ECD comprises amino acid residues numbered1-124), as in SEQ ID NO:162. A mature hCTLA-4IgG2 dimer is the form ofthe fusion protein typically used in the assays of the Examplesdescribed infra, unless stated otherwise. A DNA sequence encoding thehCTLA-4-IgG2 fusion protein, which comprises the hCTLA-4 ECD fused tothe hIgG2 Fc polypeptide, is shown in SEQ ID NO:163.

FIG. 3 represents SDS/PAGE analyses of the following proteins: molecularweight markers of various mass (kilodaltons (kDa) (lane 1); an exemplarymutant CTLA-4-Ig fusion protein of the invention based on clone D3(i.e., D3-IgG2) (lane 2); an exemplary mutant CTLA-4-Ig fusion proteinbased on clone D4 (i.e., D4-IgG2) (lane 3); and the Orencia® (Abatacept)fusion protein (lane 4) (Bristol-Myers Squibb Co., Princeton, N.J.).

FIG. 4 presents an elution profile of an exemplary mutant CTLA-4-Igfusion protein of the invention (i.e., D3-IgG2) from SEC analysis,demonstrating that mutant CTLA-4-Ig fusion proteins of the invention arehomogenous in size when purified from transiently-transfected COS cells.

FIG. 5 shows a typical Biacore™ analysis of the binding of the followingfusion proteins to hCD86-Ig: Orencia® fusion protein, LEA29Y-Ig, andD3-IgG2. The dissociation phase of the analysis begins at the timemarked by the arrow. The Orencia® fusion protein, which is composed ofthe wild-type human CTLA-4 ECD polypeptide fused to a mutant IgG1 Fcdomain polypeptide, effectively serves as a wild-type human CTLA-4-Igcontrol. A mutant CTLA-4-Ig fusion protein of the invention, such asD3-IgG2, which has a higher avidity binding to CD86-Ig than the Orencia®fusion protein has a slower rate of dissociation from CD86-Ig than theOrencia® protein.

FIG. 6 is a graphical representation of the results of PBMCproliferation inhibition assays (with anti-CD3 antibody stimulation)involving exemplary mutant CTLA-4-Ig fusion proteins of the invention(D3-04-IgG2, D3-11-IgG2, D3-12-IgG2, D3-14-IgG2). These assays show thatmutant CTLA-4-Ig fusion proteins of the invention are significantly morepotent than Orencia® and LEA29Y-Ig in inhibiting T cell proliferation invitro.

FIG. 7 is a graphical representation of CD4⁺ T cell proliferationinhibition assays (with anti-CD3 stimulation and hB7.2-dependentcostimulation) involving an exemplary set of mutant CTLA-4-Ig fusionproteins of the invention. The Orencia® and LEA29Y-Ig fusion proteinswere included as controls for comparison.

FIG. 8 is a graphical representation of PBMC proliferation inhibitionassays (with PPD antigen stimulation) involving an exemplary set ofmutant CTLA-4-Ig fusion proteins of the invention. Orencia® andLEA29Y-Ig were included as controls for comparison.

FIG. 9 is a graphical representation of one-way mixed lymphocytereaction (MLR) proliferation inhibition assays involving an exemplarymutant CTLA-4-Ig fusion protein of the invention—D3-IgG2. The Orencia®and LEA29Y-Ig fusion proteins were included as controls for comparison.

FIG. 10 is a schematic diagram showing the structure of an exemplarymutant CTLA-4-Ig fusion protein of the invention. Two identicalmonomeric mutant CTLA-4-Ig fusion proteins are shown schematically, eachcomprising a mature mutant CTLA-4 ECD fused at its C-terminus to theN-terminus of a human IgG2 Fc polypeptide. Each human IgG2 polypeptideincludes an IgG2 hinge, CH2 domain, and CH3 domain. Exemplary amino acidresidues present at the junctions between the ECD and Ig Fc polypeptidesare also shown. The amino acid residues at the junctions between thesecomponents may differ depending upon the mutant CTLA-4 ECD polypeptidesequence and/or Ig polypeptide sequence. The dimeric fusion proteinresults from the formation of at least one disulfide bond betweencysteine residues at analogous positions in the two monomers. Thecysteine (C) residues potentially involved in forming disulfide bondsbetween the two monomers are marked with asterisks. The signal peptideof each monomeric fusion protein is typically cleaved during processingand thus the secreted (mature) fusion protein typically does not includethe signal peptide sequence.

FIG. 11 is a graphical representation of CD4⁺ T cell proliferationassays (with anti-CD3 stimulation and hB7.2-dependent costimulation)involving hCTLA-4-IgG2, Orencia® and LEA29Y-Ig fusion proteins.

FIGS. 12A-12F present an alignment of the polypeptide sequence of thewild-type human CTLA-4 extracellular domain (designated in the figure as“hCTLA4ECD”), the polypeptide sequence of the LEA29Y polypeptide(designated in the figure as “LEA29YECD”), and the polypeptide sequencesof exemplary mutant CTLA-4 ECD polypeptides of the invention. The clonenames of these mutant CTLA-4 ECD polypeptides of the invention areindicated at the left. Amino acid residues that are identical to thosein the wild-type human CTLA-4 ECD are indicated with a period (.).

FIG. 13 presents a BLOSUM62 matrix.

FIGS. 14A-14D show exemplary alignments and alignment scores determinedby manual calculation for two amino acid sequences.

FIGS. 15A-15B show pharmacokinetic (PK) profiles for Orencia® fusionprotein, human CTLA-4-IgG2, and representative mutant CTLA-4-IgG2 fusionproteins of the invention administered at 1 mg/kg as a single (A)intravenous (IV) bolus or (B) subcutaneous (SC) injection in rats.

DETAILED DESCRIPTION OF THE INVENTION Definitions

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which the invention pertains.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyto refer to a polymer of nucleic acid residues (e.g.,deoxyribonucleotides or ribonucleotides) in either single- ordouble-stranded form. Unless specifically limited, the terms encompassnucleic acids containing known analogues of natural nucleotides thathave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally-occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary nucleotide sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605 2608(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes8:91 98 (1994)). The term nucleic acid or polynucleotide is usedinterchangeably with cDNA or mRNA encoded by a gene.

The term “gene” broadly refers to any nucleic acid segment (e.g., DNA)associated with a biological function. A gene may include a codingsequence and/or regulatory sequence required for their expression. Agene may also include non-expressed DNA nucleic acid segment(s) that,e.g., form recognition sequences for other protein(s) (e.g., promoter,enhancer, or other regulatory region). A gene can be obtained from avariety of sources, including cloning from a source of interest orsynthesizing from known or predicted sequence information, and mayinclude one or more sequences designed to have desired parameters.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termsapply to amino acid polymers in which one or more amino acid residue isan artificial chemical mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers andnon-naturally occurring amino acid polymers. As used herein, the termsencompass amino acid chains of any length, including full-lengthproteins (i.e., antigens), wherein the amino acid residues are linked bycovalent peptide bonds.

Numbering of a given amino acid polymer or nucleic acid polymer“corresponds to” or is “relative to” the numbering of a selected aminoacid polymer or nucleic acid polymer when the position of any givenpolymer component (e.g., amino acid, nucleotide, also referred togenerically as a “residue”) is designated by reference to the same or anequivalent position in the selected amino acid or nucleic acid polymer,rather than by the actual numerical position of the component in thegiven polymer. Thus, for example, the numbering of a given amino acidposition in a given polypeptide sequence corresponds to the same orequivalent amino acid position in a selected polypeptide sequence usedas a reference sequence.

An “equivalent position” (for example, an “equivalent amino acidposition” or “equivalent nucleic acid position” or “equivalent residueposition”) is defined herein as a position (such as an amino acidposition or nucleic acid position or residue position) of a testpolypeptide (or test polynucleotide) sequence which aligns with acorresponding position of a reference polypeptide (or referencepolynucleotide) sequence, when aligned (preferably optimally aligned)using an alignment algorithm as described herein. The equivalent aminoacid position of the test polypeptide sequence need not have the samenumerical position number as the corresponding position of the testpolypeptide. Likewise, the equivalent nucleic acid position of the testpolynucleotide sequence need not have the same numerical position numberas the corresponding position of the test polynucleotide.

A “mutant” polypeptide comprises a polypeptide sequence that differs inone or more amino acid residues from the polypeptide sequence of aparent or reference polypeptide (such as, e.g., a wild-type (WT)polypeptide sequence). In one aspect, a mutant polypeptide comprises apolypeptide sequence which differs from the polypeptide sequence of aparent or reference polypeptide in from about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 30% 40%, 50% or more ofthe total number of residues of the parent or reference polypeptidesequence. In another aspect, a mutant polypeptide comprises apolypeptide sequence that has at least about 50%, 60%, 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to the polypeptide sequence of a parent or referencepolypeptide. In another aspect, a mutant polypeptide comprises apolypeptide sequence that differs from the polypeptide sequence of aparent or reference polypeptide in from 1 to 100 or more amino acidresidues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more amino acid residues). A mutant polypeptide maycomprise a polypeptide sequence that differs from the polypeptidesequence of a parent or reference polypeptide by, e.g., the deletion,addition, or substitution of one or more amino acid residues (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore amino acid residues) of the parent or reference polypeptide, or anycombination of such deletion(s), addition(s), and/or substitution(s).The reference or parent polypeptide may itself be a mutant polypeptide.

A “mutant” nucleic acid comprises a nucleotide sequence that differs inone or more nucleic acid residues from the nucleotide sequence of aparent or reference nucleic acid (such as a WT nucleic acid). In oneaspect, a mutant nucleic acid comprises a nucleotide sequence whichdiffers from the nucleotide sequence of a parent or reference nucleicacid in from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 20%, 30% 40%, 50% or more of the total number of residuesof the parent or reference nucleotide sequence. In another aspect, amutant nucleic acid comprises a nucleotide sequence that has at leastabout 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotidesequence of a parent or reference nucleic acid. In another aspect, amutant nucleic acid comprises a nucleotide sequence that differs fromthe nucleotide sequence of a parent or reference nucleic acid in from 1to 100 or more nucleotide residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotide residues). Amutant nucleic acid may comprise a nucleotide sequence that differs fromthat of a parent or reference nucleic acid by, e.g., the deletion,addition, or substitution of one or more nucleotide residues (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore nucleotide residues) of the parent or reference nucleic acid, orany combination of such deletion(s), addition(s), and/orsubstitution(s). A mutation in a nucleic acid may also result from analternative splicing or truncation of nucleotides or errors inprocessing or cleavage of nucleotides. The reference or parent nucleicacid may itself be a mutant nucleic acid.

“Naturally occurring” as applied to an object means the object is foundin nature as distinct from being artificially produced by man.“Non-naturally occurring” as applied to an object means the object isnot naturally occurring (i.e., that the object cannot be found innature). For example, a non-naturally occurring polypeptide refers to apolypeptide that has been prepared by man, such as, for example, bybeing synthesized in vitro or prepared artificially.

A “subsequence” or “fragment” of a sequence of interest is any portionof the entire sequence, up to but not including the entire sequence ofinterest.

A nucleic acid, protein or other component is “isolated” when it ispartially or completely separated from components with which it isnormally associated (other proteins, nucleic acids, cells, syntheticreagents, etc.). On a molar basis, an isolated species is more abundantthan other species in a composition. For example, an isolated speciesmay comprise at least about 50%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% (on a molar basis) of allmacromolecular species present. Preferably, the species of interest ispurified to essential homogeneity (i.e., contaminant species cannot bedetected in the composition by conventional detection methods). Purityand homogeneity can be determined using a number of techniques wellknown in the art, such as agarose or polyacrylamide gel electrophoresisof a protein or nucleic acid sample, followed by visualization uponstaining. If desired, a high-resolution technique, such as highperformance liquid chromatography (HPLC) or a similar means can beutilized for purification of the material.

The term “purified” as applied to nucleic acids or polypeptidesgenerally denotes a nucleic acid or polypeptide that is essentially freefrom other components as determined by analytical techniques well knownin the art (e.g., a purified polypeptide or polynucleotide forms adiscrete band in an electrophoretic gel, chromatographic eluate, and/ora media subjected to density gradient centrifugation). For example, anucleic acid or polypeptide that gives rise to essentially one band inan electrophoretic gel is “purified.” A purified nucleic acid orpolypeptide is at least about 50% pure, usually at least about 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%,99.7%, 99.8% or more pure (e.g., percent by weight on a molar basis).

In a related sense, the invention provides methods of enrichingcompositions for one or more molecules of the invention, such as one ormore polypeptides or polynucleotides of the invention. A composition isenriched for a molecule when there is a substantial increase in theconcentration of the molecule after application of a purification orenrichment technique. A substantially pure polypeptide or polynucleotidewill typically comprise at least about 55%, 60%, 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, 99%, 99.5% or more by weight (ona molar basis) of all macromolecular species in a particularcomposition.

A nucleic acid or polypeptide is “recombinant” when it is artificial orengineered, or derived from an artificial or engineered protein ornucleic acid.

The term “recombinant” when used with reference to a cell typicallyindicates that the cell replicates a heterologous nucleic acid orexpresses a polypeptide encoded by a heterologous nucleic acid.Recombinant cells can comprise genes that are not found within thenative (non-recombinant) form of the cell. Recombinant cells alsoinclude those that comprise genes that are found in the native form ofthe cell, but are modified and re-introduced into the cell by artificialmeans. The term also encompasses cells that comprise a nucleic acidendogenous to the cell that has been modified without removing thenucleic acid from the cell; such modifications include those obtained bygene replacement, site-specific mutation, and related techniques knownto those of ordinary skill in the art. Recombinant DNA technologyincludes techniques for the production of recombinant DNA in vitro andtransfer of the recombinant DNA into cells where it may be expressed orpropagated, thereby producing a recombinant polypeptide.

A “recombinant expression cassette” or simply an “expression cassette”is a nucleic acid construct, generated recombinantly or synthetically,with nucleic acid elements that are capable of effecting expression of astructural gene in hosts compatible with such sequences. Expressioncassettes include at least promoters and optionally transcriptiontermination signals. Typically, the recombinant expression cassetteincludes a nucleic acid to be transcribed (e.g., a nucleic acid encodinga desired polypeptide) and a promoter. Additional factors necessary orhelpful in effecting expression may also be used as described herein.For example, an expression cassette can also include nucleotidesequences that encode a signal sequence that directs secretion of anexpressed protein from the host cell. Transcription termination signals,enhancers, and other nucleic acid sequences that influence geneexpression, can also be included in an expression cassette.

An “exogenous” nucleic acid,” “exogenous DNA segment,” “heterologoussequence,” or “heterologous nucleic acid,” as used herein, is one thatoriginates from a source foreign to the particular host cell, or, iffrom the same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell, but has been modified. Modification of aheterologous sequence in the applications described herein typicallyoccurs through the use of directed molecular evolution methods. Thus,the terms refer to a DNA segment which is foreign or heterologous to thecell, or homologous to the cell but in a position within the host cellnucleic acid in which the element is not ordinarily found. Exogenousnucleic acids or exogenous DNA are expressed to yield exogenouspolypeptides.

A “vector” may be any agent that is able to deliver or maintain anucleic acid in a host cell and includes, for example, but is notlimited to, plasmids (e.g., DNA plasmids), naked nucleic acids, viralvectors, viruses, nucleic acids complexed with one or more polypeptideor other molecules, as well as nucleic acids immobilized onto solidphase particles. Vectors are described in detail below. A vector can beuseful as an agent for delivering or maintaining an exogenous geneand/or protein in a host cell. A vector may be capable of transducing,transfecting, or transforming a cell, thereby causing the cell toreplicate or express nucleic acids and/or proteins other than thosenative to the cell or in a manner not native to the cell. A vector mayinclude materials to aid in achieving entry of a nucleic acid into thecell, such as a viral particle, liposome, protein coating, or the like.Any method of transferring a nucleic acid into the cell may be used;unless otherwise indicated, the term vector does not imply anyparticular method of delivering a nucleic acid into a cell or imply thatany particular cell type is the subject of transduction. The presentinvention is not limited to any specific vector for delivery ormaintenance of any nucleic acid of the invention, including, e.g., anucleic acid encoding a mutant CTLA-4 polypeptide of the invention or afragment thereof (e.g., mutant CTLA-4 ECD) that binds CD80 and/or CD86or a fragment thereof (e.g., a CD80 ECD or CD86 ECD).

The term “expression vector” typically refers to a nucleic acidconstruct or sequence, generated recombinantly or synthetically, with aseries of specific nucleic acid elements that permit transcription of aparticular nucleic acid in a host cell. The expression vector typicallyincludes a nucleic acid to be transcribed operably linked to a promoter.The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and/or secretion.

A “signal peptide” is a peptide (or amino acid) sequence that typicallyprecedes a polypeptide of interest and is translated in conjunction withthe polypeptide and directs or facilitates the polypeptide to thesecretory system. A signal peptide is typically covalently attached orfused to the amino terminus of the polypeptide of interest andfacilitates secretion of the polypeptide of interest from a host cell.The signal peptide is typically cleaved from the polypeptide of interestfollowing translation.

The term “encoding” refers to the ability of a nucleotide sequence tocode for one or more amino acids. The term does not require a start orstop codon. An amino acid sequence can be encoded in any one of sixdifferent reading frames provided by a polynucleotide sequence and itscomplement.

The term “control sequence” is defined herein to include all components,which are necessary or advantageous for the expression of a polypeptideof the present invention. Each control sequence may be native or foreignto the nucleotide sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, a control sequence includes apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

The term “coding sequence” refers to a nucleotide sequence that directlyspecifies the amino acid sequence of its protein product. The boundariesof the coding sequence are generally determined by an open reading frame(ORF), which may begin with the ATG start codon.

A nucleic acid is “operably linked” with another nucleic acid sequencewhen it is placed into a functional relationship with another nucleicacid sequence. For instance, a promoter or enhancer is operably linkedto a coding sequence if it directs transcription of the coding sequence.Operably linked means that the DNA sequences being linked are typicallycontiguous and, where necessary to join two protein coding regions,contiguous and in reading frame. However, since enhancers generallyfunction when separated from the promoter by several kilobases andintronic sequences may be of variable lengths, some polynucleotideelements may be operably linked but not contiguous.

A “host cell” is any cell that is susceptible to transformation with anucleic acid.

“Substantially the entire length of a polynucleotide sequence” or“substantially the entire length of a polypeptide sequence” refers to atleast about 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more of the length of apolynucleotide sequence or polypeptide sequence, respectively.

An “antigen” refers to a substance that reacts with the product(s) of animmune response stimulated by a specific immunogen. See, e.g., JULIUSCRUSE ET AL., ATLAS OF IMMUNOLOGY 60 (1999); RICHARD COICO ET AL.,IMMUNOLOGY: A SHORT COURSE 27-30 (5^(th) ed. 2003). An immune responsemay comprise a humoral response and/or a cell-mediated immune response(e.g., cytotoxic T lymphocytes (CTLs)). Products of an immune responsemay include antibodies and/or CTLs. Antigens are typicallymacromolecules (e.g., polypeptides, nucleic acids, complexcarbohydrates, phospholipids, polysaccharides) that are foreign to thehost; that portion of the antigen known as the antigenic determinantreacts with (e.g., binds to) the product(s) of the immune response, suchas an antibody or a specific T cell receptor on a T lymphocyte. Anantigen may, but not necessarily, induce an immune response as well asreact with the product(s) of the immune response. “Antigenicity” refersthe state or property of being antigenic—i.e., having the properties ofan antigen. Specificity of an antigen may be shown in the relation of anantigen to its antibody or vice versa; an antigen typically reacts in ahighly specific fashion with its corresponding antibody and not with thesame degree of specificity with other antibodies evoked by theimmunogen. An “antigenic amount” is an amount of an antigen thatdetectably reacts with the product(s) of an immune response stimulatedby a specific immunogen.

An “immunogen” is a substance that is capable of inducing an immuneresponse rather than immunological tolerance. See, e.g., JULIUS CRUSE ETAL., supra at 60-61; RICHARD COICO, supra at 27-30. Immunogens alsoreacts with (e.g., bind) the product(s) of the induced immune responsethat has or have been specifically induced against them. Thus, allimmunogens are antigens. “Immunogenicity” refers the state or propertyof being immunogenic—i.e., having the properties of an immunogen. An“immunogenic amount” is an amount of an immunogen that is effective toinduce a detectable an immune response. An immunogen may elicit a strongimmune response in a subject, such as at least partial or completeprotective immunity to at least one pathogen.

An “immunomodulator” or “immunomodulatory” molecule, such as animmunomodulatory polypeptide or nucleic acid, modulates an immuneresponse. By “modulation” or “modulating” an immune response is intendedthat the immune response is altered. For example, “modulation” of or“modulating” an immune response in a subject generally means that animmune response is stimulated, induced, inhibited, decreased,suppressed, increased, enhanced, or otherwise altered in the subject.Such modulation of an immune response can be assessed by means known tothose skilled in the art, including those described below. An“immunosuppressor” or “immunosuppressant” is a molecule, such as apolypeptide or nucleic acid, which suppresses an immune response.

As used herein, an “antibody” (abbreviated “Ab”) refers to animmunoglobulin protein (abbreviated “Ig”), whether natural or wholly orpartially synthetically produced. The term includes all derivativesthereof that maintain specific binding ability to an antigen. The termalso covers any protein having a binding domain that is homologous orlargely homologous to an immunoglobulin binding domain. Such proteinsmay be derived from natural sources, or partly or wholly syntheticallyproduced. An antibody may be monoclonal or polyclonal. An antibody maybe a member of any immunoglobulin class, including any of the five humanclasses: IgA (which includes subclasses IgA1 and IgA2), IgD, IgE, IgG(which includes subclasses IgG1, IgG2, IgG3, and IgG4), and IgM.Antibodies comprise paired heavy and light polypeptide chains, and eachsuch chain is composed of individual immunoglobulin domains. Each chainincludes a constant (C) region and a variable (V) region. A typicalantibody structural unit comprises a tetramer. Each tetramer is composedof two identical pairs of polypeptide chains, each pair having one“light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Heavychains exist in five major types (γ, μ, δ, α, and ε) depending on theantibody class and contain about 450-600 amino acid residues. Lightchains are of two major types (λ and κ) and contain about 230 amino acidresidues. As an example, an IgG antibody is a tetrameric proteincomprising two heavy chains and two light chains. Each IgG heavy chaincontains four immunoglobulin domains linked in the following order fromthe N-terminus to the C-terminus: V_(H)-C_(H)1-C_(H)2-C_(H)3 (alsosometimes abbreviated as VH-CH1-CH2-CH3). These abbreviations refer tothe heavy chain variable domain, heavy chain constant domain 1, heavychain constant domain 2, and heavy chain constant domain 3),respectively. A heavy chain may also be referred to by antibody class,such as, e.g., Cγ1, which represents the first constant domain of thegamma (γ) heavy chain of IgG antibody. Each IgG light chain comprisestwo immunoglobulin domains linked in the following order from N- toC-terminus: V_(H)-C_(L), wherein V_(H) and C_(L) refer to the lightchain variable domain and light chain constant domain, respectively.

The variable region of an antibody, which typically comprises about 100to 110 or more amino acids at the N-terminus of each polypeptide chain,includes the antigen binding determinants and thus is primarilyresponsible for antigen recognition and specificity. The greatest degreeof amino acid sequence variability between antibodies is found in thevariable region. Most sequence variability occurs in the complementaritydetermining regions (CDRs) located in the variable region. There are atotal of six CDRs, three CDRs in each heavy chain and three CDRs in eachlight chain, which together form the antigen-binding site. The heavychain CDRs are designated as V_(H) CDR1, V_(H) CDR2, and V_(H) CDR3while the light chain CDRs are designated as V_(L) CDR1, V_(L) CDR2, andV_(L) CDR3. The region located outside the CDRs is termed the framework(FR) region. Framework regions of different antibodies may vary in aminoacid residues, but the degree of amino acid variability is not nearly asgreat as that which exists between the variable regions of differentantibodies. In many instances, the framework regions provide a stable orconstant scaffold for the amino acid diversity presented by the CDRs.

The term “antibody fragment” refers to any derivative of an antibodythat is less than full-length. Examples of antibody fragments include,but are not limited to, e.g., the antigen binding fragment (Fab)containing V_(H)-C_(H)1 and V_(H)-C_(L), the variable fragment (Fv)containing V_(H) and V_(L), the single chain variable fragment (scFv)containing V_(H) and V_(L) linked together in one chain, as well asother V region fragments, such as Fab′, F(ab)₂, F(ab′)₂, dsFv diabody,Fc, and Fd polypeptide fragments. See Scott, T. A. and Mercer, E. I.,CONCISE ENCYCLOPEDIA: BIOCHEMISTRY AND MOLECULAR BIOLOGY (de Gruyter, 3ded. 1997), and Watson, J. D. et al., RECOMBINANT DNA (2d ed. 1992)(hereinafter “Watson”).

An antibody fragment may be produced by any means known in the art. Forinstance, the antibody fragment may be enzymatically or chemicallyproduced by fragmentation of an intact antibody or it may berecombinantly produced from a gene encoding the partial antibodysequence. For example, fragments of antibodies can be produced bydigestion with a peptidase. For example, pepsin digests an antibodybelow the disulfide linkages in the hinge region to produce F(ab′)₂, adimer of a Fab fragment which itself is a light chain joined toV_(H)-C_(H)1 by a disulfide bond. The F(ab′)₂ may be reduced under mildconditions to break the disulfide linkage in the hinge region therebyconverting the (Fab′)₂ dimer into an Fab′ monomer. The Fab′ monomer isessentially a Fab fragment with part of the hinge region. SeeFUNDAMENTAL IMMUNOLOGY, W. E. Paul, ed., Raven Press, N.Y. (1993) for amore detailed description of antibodies and antibody fragments. Whilevarious antibody fragments are defined in terms of the digestion of anintact antibody, one of skill will appreciate that such Fab′ fragmentsmay be synthesized de novo either chemically or by utilizing recombinantDNA methodology. Thus, the term also includes antibody fragments eitherproduced by the modification of whole antibodies or synthesized de novousing recombinant DNA methodologies.

Alternatively, the antibody fragment may be wholly or partiallysynthetically produced. The antibody fragment may optionally be a singlechain antibody fragment. Alternatively, the fragment may comprisemultiple chains that are linked together, for instance, by disulfidelinkages. The fragment may also optionally be a multimolecular complex.A functional antibody fragment will typically comprise at least about 50amino acids and more typically will comprise at least about 200 aminoacids.

An Fc region or domain of an immunoglobulin or antibody molecule (alsotermed an Ig Fc polypeptide or Fc polypeptide) corresponds largely tothe constant region of the immunoglobulin heavy chain, and isresponsible for various functions, including the antibody's effectorfunction(s). For example, the Ig Fc region of IgG molecule comprises theimmunoglobulin domains CH2 and CH3 and the N-terminal hinge regionleading into CH2. The hinge region is a portion of the heavy chainbetween Fc and CH1 containing the inter-heavy chain disulfide binds andgives flexibility to the antibody molecule. The constant domains of theFc region interact with cells of the immune system. Fc receptors areproteins that bind the Fc region of antibodies. One significant familyof Fc receptors for the IgG antibody class includes the Fc gammareceptors (FcγR). The binding of antibodies to Fc receptors on cellsmediates a number of antibody functions. Different IgG subclassesexhibit different affinities for Fc gamma receptors. In general, IgG1and IgG3 bind to the receptors with a greater affinity than IgG2 andIgG4. Fc receptors are expressed on a variety of cells, including, e.g.,B cells, monocytes, dendritic cells, neutrophils, and certainlymphocytes. Binding of an Ig Fc to its receptor brings these effectorcells to sites of the bound antigen, resulting ultimately in signalingand immune responses, including B cell activation, inflammatoryresponses, cytotoxic responses, and phagocytic responses.

An Ig Fc fusion is a molecule comprising one or more polypeptides (orone or more small molecules) operably linked to an Fc region of animmunoglobulin or antibody. See, e.g., Chamow et al., 1996, TrendsBiotechnol. 14:52-60. Accordingly, an Ig Fc fusion protein is a moleculecomprising one or more polypeptides operably linked to an Ig Fc region.An Ig Fc fusion protein may comprise, for example, the Fc region of anantibody (which facilitates effector functions and pharmacokinetics) andthe binding region or binding domain of a receptor protein or ligandprotein or other protein or fragment thereof. The binding region orbinding domain mediates recognition of the target receptor or ligand(comparable to that of antibody variable region of an antibody for anantigen). An Ig Fc region may be linked indirectly or directly to one ormore polypeptides or small molecules (fusion partners). Various linkersknown in the art and as described in greater detail below can be used tolink an Ig Fc to a fusion partner to generate an Ig Fc fusion. An Ig Fcfusion protein typically comprises an Ig Fc region covalently linkeddirectly or indirectly to at least one polypeptide, which polypeptidetypically binds a target ligand or receptor.

Monoclonal or polyclonal antibodies can be prepared any technique knownin the art can be used (see, e.g., Kohler & Milstein, Nature 256:495 497(1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp.77 96 in Monoclonal Antibodies and Cancer Therapy (1985)). Techniquesfor the production of single chain antibodies (U.S. Pat. No. 4,946,778)can be adapted to produce antibodies to polypeptides of the invention.In addition, transgenic mice or other organisms, including mammals, maybe used to express humanized antibodies. Phage display technology can beused to identify antibodies and heteromeric Fab fragments thatspecifically bind to selected antigens (see, e.g., McCafferty et al.,Nature 348:552 554 (1990); Marks et al., Biotechnology 10:779 783(1992)).

The term “epitope” refers to an antigenic determinant capable ofspecific binding to a part of an antibody. Epitopes usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific 3-dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents.

A “specific binding affinity” between two molecules, e.g., a ligand anda receptor, means a preferential binding of one molecule for another.The binding of molecules is typically considered specific if theequilibrium binding association constant (e.g., K_(A)) is about 1×10²M⁻¹ to about 1×10¹³ M⁻¹ or greater, including about 10⁴ to 10¹³ M⁻¹,about 10⁶ to 10¹² M⁻¹, about 10⁸ M⁻¹ to 10¹¹ M⁻¹ or about 10⁸ to 10¹⁰M⁻¹. Values of K_(A) for the binding interaction between an antigen andan antibody typically range from about 10⁵ M⁻¹ to about 10¹² M⁻¹,usually about 10⁷ M⁻¹ to about 10¹¹ M⁻¹, and often about 10⁸ M⁻¹ toabout 10¹⁰ M⁻¹. K_(A) (M⁻¹) is determined by calculating k_(a)/k_(d),where k_(a) is the association rate constant and k_(d) is thedisassociation rate constant. The units of k_(a) and k_(d) are M⁻¹ s⁻¹and s⁻¹, respectively. The equilibrium dissociation constant, K_(D), isthe reciprocal of K_(A). K_(D)=k_(d)/k_(a). For the reaction A+B<=>AB(representing a single ligand binding to a single protein of interest(e.g., receptor)), K_(D) is equal to ([A]·[B])/[AB]. Non-limitingexamples of well-known techniques for measuring binding affinitiesand/or avidities of molecules include, e.g., Biacore™ technology (GEHealthcare) as discussed elsewhere herein, isothermal titrationmicrocalorimetry (MicroCal LLC, Northampton, Mass. USA), ELISA, andfluorescence activated cell sorting (FACS) methods. For example, FACS orother sorting methods may be used to select for populations of molecules(such as for example, cell surface-displayed ligands) that specificallybind to the associated binding pair member (such as a receptor, e.g., asoluble receptor). Ligand-receptor complexes may be detected and sortede.g., by fluorescence (e.g., by reacting the complex with a fluorescentantibody that recognizes the complex). Molecules of interest that bindan associated binding pair member (e.g., receptor) are pooled andre-sorted in the presence of lower concentrations of receptor. Byperforming multiple rounds sorting in the presence of decreasingconcentrations of receptor (an exemplary concentration range being onthe order of 10⁻⁶ M down to 10⁻¹³ M, i.e., 1 micromolar (μM) down to 1nanomolar (nM), or less (e.g., 10⁻¹¹ M or 10⁻¹²M), depending on thenature of the ligand-receptor interaction), populations of the moleculeof interest exhibiting specific binding affinity for the receptor may beisolated.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein, refers to a binding reaction that is determinative of thepresence of the protein in a heterogeneous population of proteins andother biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. A variety of immunoassay formatsmay be used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual(1988), for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity). Usually, a specific orselective reaction will be at least twice background signal or noise andmore typically more than 10 to 100 times background.

The term “cytokine” includes, e.g., but is not limited to, interleukins,interferons (IFN), chemokines, hematopoietic growth factors, tumornecrosis factors (TNF), and transforming growth factors. In general,these are small molecular weight proteins that regulate maturation,activation, proliferation, and differentiation of cells of the immunesystem.

The term “screening” describes, in general, a process that identifiesoptimal molecules of the present invention, such as, e.g., includingpolypeptides of the invention, and related fusion proteins comprisingthe same, and nucleic acids encoding all such molecules. Severalproperties of the respective molecules can be used in selection andscreening, for example, an ability of a respective molecule to induce oralter a desired immune response in a test system or in an in vitro, exvivo, or in vivo application. “Selection” is a form of screening inwhich identification and physical separation are achieved simultaneouslyby expression of a selection marker, which, in some geneticcircumstances, allows cells expressing the marker to survive while othercells die (or vice versa). Screening markers include, for example,luciferase, beta-galactosidase and green fluorescent protein, reactionsubstrates, and the like. Selection markers include drug and toxinresistance genes, and the like. Another mode of selection involvesphysical sorting based on a detectable event, such as binding of aligand to a receptor, reaction of a substrate with an enzyme, or anyother physical process which can generate a detectable signal eitherdirectly (e.g., by utilizing a chromogenic substrate or ligand) orindirectly (e.g., by reacting with a chromogenic secondary antibody).Selection by physical sorting can by accomplished by a variety ofmethods, including, but not limited to, e.g., y FACS in whole cell ormicrodroplet formats.

Because of limitations in studying primary immune responses in vitro, invivo studies are particularly useful screening methods. In some suchstudies, a polynucleotide or polypeptide of the invention is firstintroduced to a test subject (e.g., a mammal, such as an animal), and aninduced immune response is subsequently studied by analyzing the type ofimmune response in the immunized animal (e.g., antibody production inthe immunized animal's serum, proliferation of T cells), or by studyingthe quality or strength of the induced immune response in the immunizedanimal (e.g., induced antibody titer level).

The term “subject” as used herein includes, but is not limited to, anorganism or animal, including mammals and non-mammals. A mammalincludes, e.g., but is not limited to, a human, non-human primate (e.g.,baboon, orangutan, monkey, gorilla), mouse, dog, pig, cow, goat, cat,rabbit, rat, guinea pig, hamster, horse, sheep, or other non-humanmammal. A non-mammal includes, e.g., but is not limited to, anon-mammalian invertebrate and non-mammalian vertebrate, such as a bird(e.g., a chicken or duck) or a fish.

The term “pharmaceutical composition” refers to a composition suitablefor pharmaceutical use in a subject, including an animal or human. Apharmaceutical composition typically comprises an effective amount of anactive agent and a carrier, excipient, or diluent. The carrier,excipient, or diluent is typically a pharmaceutically acceptablecarrier, excipient or diluent, respectively.

The term “effective amount” refers to a dosage (or dose) or amount of asubstance sufficient to produce a desired result. The desired result maycomprise an objective or subjective improvement in the recipient of thedosage or amount. For example, the desired result may comprise ameasurable, detectable or testable induction, promotion, enhancement ormodulation of an immune response in a subject to whom a dosage or amountof a particular antigen or immunogen (or composition thereof) has beenadministered. A dosage (or dose) or amount of an immunogen sufficient toproduce such result can be described as an “immunogenic” dosage (ordose) or amount.

A “prophylactic treatment” is a treatment administered to a subject whodoes not display signs or symptoms of, or displays only early signs orsymptoms of, a disease, pathology, or disorder, such that treatment isadministered for the purpose of preventing or decreasing the risk ofdeveloping the disease, pathology, or disorder. A prophylactic treatmentfunctions as a preventative treatment against a disease, pathology, ordisorder, or as a treatment that inhibits or reduces further developmentor enhancement of a disease, pathology or disorder. A “prophylacticactivity” is an activity of an agent that, when administered to asubject who does not display signs or symptoms of, or who displays onlyearly signs or symptoms of, a pathology, disease, or disorder, preventsor decreases the risk of the subject developing the pathology, disease,or disorder. A “prophylactically useful” agent (e.g., nucleic acid orpolypeptide) refers to an agent that is useful in preventing developmentof a disease, pathology, or disorder, or useful in inhibiting orreducing further development or enhancement of a disease, pathology ordisorder.

A “therapeutic treatment” is a treatment administered to a subject whodisplays symptoms or signs of pathology, disease, or disorder, in whichtreatment is administered to the subject for the purpose of diminishingor eliminating those signs or symptoms. A “therapeutic activity” is anactivity of an agent that eliminates or diminishes signs or symptoms ofpathology, disease or disorder when administered to a subject sufferingfrom such signs or symptoms. A “therapeutically useful” agent means theagent is useful in decreasing, treating, or eliminating signs orsymptoms of a disease, pathology, or disorder.

Generally, the nomenclature used herein and many of the laboratoryprocedures in cell culture, molecular genetics, molecular biology,nucleic acid chemistry, and protein chemistry described below are wellknown and commonly employed by those of ordinary skill in the art.Standard techniques, such as described in Sambrook et al., MolecularCloning—A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989 (hereinafter “Sambrook”) andCURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc. (1994, supplemented through 1999)(hereinafter “Ausubel”), are used for recombinant nucleic acid methods,nucleic acid synthesis, cell culture methods, and transgeneincorporation, e.g., electroporation, injection, gene gun, impressingthrough the skin, and lipofection. Generally, oligonucleotide synthesisand purification steps are performed according to specifications. Thetechniques and procedures are generally performed according toconventional methods in the art and various general references that areprovided throughout this document. The procedures therein are believedto be well known to those of ordinary skill in the art and are providedfor the convenience of the reader.

Various additional terms are defined or otherwise characterized herein.

Molecules and Methods of the Invention

The present invention provides molecules and methods for treatingdiseases, disorders, and conditions of the immune system, including,e.g., those in which modulation of the immune system (e.g., T-celldependent immune responses) is desirable. Molecules of the invention(e.g., polypeptides of the invention, conjugates of the invention,soluble fusion proteins of the invention, nucleic acids encoding suchpolypeptides or fusion proteins) are useful for the treatment of immunesystem diseases, disorders, and conditions in which immunosuppression isdesirable, including, e.g., but not limited to, the treatment ofautoimmune diseases, disorders, and conditions, immunoproliferativediseases, graft-related disorders, and treatment methods involvingtissue, cell, organ, or graft transplantation from a donor to arecipient where suppression of an immune response in the recipientagainst the donor tissue, cell, organ, or graft is desirable.

In one aspect, the invention provides novel mutant CTLA-4 moleculeshaving improved properties compared to a CTLA-4 molecule, such as thewild-type human CTLA-4 polypeptide (“hCTLA-4”) or a fragment thereofthat binds CD80 and/or CD86, such as the extracellular domain of humanCTLA-4 (“hCTLA-4 ECD”). As discussed in greater detail below, a varietyof mutagenesis and screening strategies were used to make and identifynovel mutant CTLA-4 molecules that bind CD80 and/or CD86. In particular,such strategies were used to make and identify CTLA-4 mutant moleculeshaving improved binding avidities for CD80 (B7-1) and/or CD86 (B7-2), ascompared to human CTLA-4 (“hCTLA-4”), and/or having improved bindingaffinities for CD80 and/or CD86, as compared to the hCTLA-4 ECD. MutantCTLA-4 molecules of the invention that bind endogenous CD80 and/or CD86ligands expressed on antigen-presenting cells effectively inhibit orblock the interaction of these ligands with the endogenous CD28receptor, which is expressed on the surface of T cells. As a result, thecostimulatory signal critical for T cell activation provided by theinteraction of the T cell surface receptor CD28 with the B7 molecules(i.e., CD80 and CD86) is inhibited or blocked. Such T cells are notoptimally activated and have reduced capacities for proliferation.

In instances where signaling between a CD80 or CD86 ligand and a CD28receptor is blocked, T cells are not optimally stimulated to becomeactive and thus are not optimally induced to proliferate. Similarly, ininstances where the signaling between a CD80 or CD86 ligand and a CD28receptor is inhibited, activation and proliferation of T cells isinhibited. In one aspect, the invention provides mutant CTLA-4 moleculesthat function as antagonists to CTLA-4 signaling. In another aspect, theinvention provides mutant CTLA-4 molecules that function as antagoniststo CD28 signaling, thereby suppressing or blocking T cell-dependentimmune responses; such molecules function as immunosuppressive agents.In yet another aspect, the invention provides mutant CTLA-4 moleculesthat bind both CD80 and CD86, but which have higher binding avidity forCD86 than for CD80, and therefore inhibit CD86-dependent costimulationto a greater extent than CD80-dependent costimulation. All such mutantCTLA-4 molecules of the invention are expected to be useful for thetreatment of diseases, disorders, or conditions in whichimmunosuppression is desirable or would be of benefit.

A human CTLA-4-Ig fusion protein and a specific mutant CTLA-4-Ig fusionprotein—both developed by Bristol-Myers Squibb Co. (Princeton,N.J.)—have been shown to be effective in treating certain immune-relateddiseases or conditions. The Orencia® fusion protein (also known asAbatacept (“ABA”)) (Bristol-Myers Squibb Co. (Princeton, N.J.)) is asoluble recombinant dimeric fusion protein consisting of two identicalmonomeric immunoglobulin (Ig) fusion proteins covalently linked togetherby a disulfide bond formed between a cysteine residue present in eachmonomeric fusion protein. ORENCIA is a registered trademark ofBristol-Myers Squibb Company. Each monomeric Ig fusion protein of theOrencia® dimer consists of the extracellular domain of human CTLA-4 (SEQID NO:159) fused at its C-terminus to the N-terminus of a specificmutant IgG1 Fc polypeptide (SEQ ID NO:186). The complete polypeptidesequence of each such monomeric fusion protein is shown in SEQ IDNO:164. The Orencia® dimer is produced in a mammalian expression systemand has an apparent molecule weight of 92 kDa. It is believed that thetwo monomeric Ig fusion proteins of the Orencia® dimer are covalentlylinked together by a single disulfide bond formed between the cysteineresidue at position 120 of each hCTLA-4-mutant IgG1 monomer and that nodisulfide bonds are formed between the two mutant IgG1 Fc polypeptides.

The Orencia® dimer is a selective costimulation modulator that inhibitsT cell activation by binding to CD80 and CD86 and thus blockinginteraction with CD28. The Orencia® dimer is currently approved for thetreatment of human adults suffering from moderate to severe rheumatoidarthritis (RA). Additional information about the Orencia® dimer and itsclinical indications and effectiveness is provided on the worldwide webat orencia.com and bms.com.

As noted above, each fusion protein monomer of the Orencia® dimercontains a human CTLA-4 extracellular domain. Human CTLA-4 is a membraneprotein that is transiently expressed on T cells. The full-lengthprotein sequence of WT full-length hCTLA-4 is shown in SEQ ID NO:160,and a nucleic acid sequence encoding WT full-length hCTLA-4 is shown inSEQ ID NO:194. Human CTLA-4 includes a signal peptide (SP),extracellular domain (ECD), transmembrane domain (TD), and cytoplasmicdomain (CD, covalently linked together in that order (e.g., theC-terminus of the SP is covalently linked to the N-terminus of the ECD,the C-terminus of the ECD is covalently linked to the N-terminus of theTD, and the C-terminus of the TD is covalently linked to the N-terminusof the CD). The WT hCTLA-4 ECD polypeptide typically comprises residues38-161 of the full-length hCTLA-4 protein sequence (SEQ ID NO:160) andtypically is 124 amino acid residues in length. This hCTLA-4 ECDpolypeptide sequence is shown in SEQ ID NO:159. The signal peptide (SP)of the full-length hCTLA-4 protein, which typically comprises amino acidresidues 1-35 or 1-37 of SEQ ID NO:160, is cleaved during processing.See, e.g., Harper et al., J. Immunol. 147(3):1037-1044 (1991). The humanCTLA-4 signal peptide sequence comprising amino acid residues 1-35 or1-37 of the hCTLA-4 protein is shown in SEQ ID NO:182 or SEQ ID NO:216,respectively. Whether the signal peptide sequence is that shown in SEQID NO:182 or SEQ ID NO:216, when the signal peptide is cleaved, themature hCTLA-4 protein typically begins with the methionine residue atamino acid position 38 of the full-length hCTLA-4 protein sequence shownin SEQ ID NO:160. Thus, even if the hCTLA-4 signal peptide sequence isthat of SEQ ID NO:182, which comprises amino acid residues 1-35 of thehCTLA-4 protein, the resulting mature secreted hCTLA-4 protein beginswith the methionine that is at position 38 of the full-length hCTLA-4protein. The lysine (K) and alanine (A) residues at positions 36 and 37,respectively, of the full-length hCTLA-4 protein are not present in themature hCTLA-4 protein and are believed to be cleaved from the maturehCTLA-4 protein during processing. The amino acid residues of the maturehCTLA-4 protein sequence thus are typically numbered beginning with themethionine residue present at position 38 of the full-length hCTLA-4protein as the first amino acid (i.e., occupying position 1);accordingly, the histidine residue occupies amino acid position 2 in themature hCTLA-4 protein, etc. Each monomer of the Orencia® dimer includesthe hCTLA-4 ECD polypeptide sequence shown in SEQ ID NO:159. In thefull-length WT hCTLA-4 protein, the signal peptide comprises amino acidresidues 1-37, the extracellular domain (ECD) comprises amino acidresidues 38-161, the transmembrane domain (TD) comprises amino acidresidues 162-182, and the cytoplasmic domain (CD) comprises amino acidresidues 183-223 of SEQ ID NO:160. The mature domain (MD) of the hCTLA-4protein typically comprises amino acid residues 36-223, or in someinstances, amino acid residues 37-223 or 38-223 of SEQ ID NO:160.

The nucleic acid of SEQ ID NO:194 comprises a nucleic acid sequenceencoding signal peptide sequence (nucleotide residues 1-111), a nucleicacid sequence encoding hCTLA-4 ECD (nucleotide residues 112-483), anucleic acid sequence encoding the hCTLA-4 transmembrane and cytoplasmicdomains (nucleotide residues 484-669); the last 3 C-terminal nucleotidesare the TGA stop codon.

Belatacept (also known as “LEA29Y-Ig,” “LEA-Ig,” or “A29YL104E-Ig”)(Bristol-Myers Squibb Co. (Princeton, N.J.)) is a soluble recombinantdimeric protein composed of two identical Ig fusion proteins covalentlylinked together by a disulfide bond formed between a cysteine residue ineach monomeric fusion protein. Each monomeric fusion protein is composedof a mutant CTLA-4 extracellular domain polypeptide fused at itsC-terminus to the N-terminus of a specific mutant IgG1 polypeptide. Thepolypeptide sequence of the mutant CTLA-4 ECD differs from thepolypeptide sequence of WT human CTLA-4 ECD by two mutations,specifically a substitution of a tyrosine for the alanine at position 29(abbreviated as the substitution A29Y) and a substitution of a glutaminefor the leucine at position 104 (abbreviated as the substitution L104E),wherein amino acid residues in the human CTLA-4 ECD are numbered withthe methionine at the N-terminus representing the amino acid atposition 1. Each monomer of Belatacept includes the mutant IgG1 Fcpolypeptide sequence shown in SEQ ID NO:186; this mutant IgG1 Fcpolypeptide is identical to the mutant IgG1 Fc polypeptide included inthe Orencia® fusion protein. Belatacept monomeric fusion protein thusdiffers from each Orencia® monomeric fusion protein by two amino acids.The polypeptide sequence of each such monomeric fusion protein inBelatacept is shown in SEQ ID NO:166. The name “LEA29Y-Ig” thus reflectsthe fact that each monomeric fusion protein of the Belatacept dimer iscomposed of a mutant CTLA-4 ECD which differs from the human CTLA-4 ECDpolypeptide sequence by two the mutations L104E and A29Y. Belatacept hasbeen shown to bind CD86 about 4 times more avidly and to bind CD80 about2 times more avidly than the Orencia® dimer (Larson et al., Amer. J.Transplant. 5:443-453, 444 (2005). Belatacept has been shown to be up toabout 10 times more potent than the Orencia® dimer in inhibiting T cellactivation in vitro and to have improved in vivo immunosuppressivepotency compared to the Orencia® protein as shown by its increasedability to inhibit T cell-dependent antibody responses and its improvedprolongation of renal allograft survival in clinical trials involvingnon-human primates. Id. Additional information about Belatacept and itsclinical indications and effectiveness is provided on the worldwide webat bms.com.

In one aspect, the invention provides mutant CTLA-4 molecules, includingnovel soluble recombinant mutant CTLA-4-Ig fusion proteins describedherein, which have a binding avidity for CD86 that is greater than thebinding avidity of the Orencia® dimer (dimeric hCTLA-4-Ig) for CD86. Theinvention also provides mutant CTLA-4 molecules, including novel solublerecombinant dimeric mutant CTLA-4-Ig fusion proteins, which have abinding avidity for CD80 that is about equal to or greater than thebinding avidity of the Orencia® dimer for CD80. In yet another aspect,the invention provides mutant CTLA-4 molecules, including novel solublerecombinant mutant CTLA-4-Ig fusion proteins, which have a greaterability to suppress one or more immune responses (e.g., T cell-dependentimmune responses) than Orencia® (Abatacept). Mutant CTLA-4 molecules ofthe invention having one or more improved properties compared to theOrencia® dimer are expected to more potent and thus more effective,useful, and advantageous than the Orencia® dimer in treating diseases,disorders, or conditions in which immunosuppression is desirable,including those diseases, disorders, or conditions for which theOrencia® dimer is approved and/or has been shown to provide clinicalbenefit, such as autoimmune diseases, including, e.g., rheumatoidarthritis and psoriasis.

In another aspect, the invention provides mutant CTLA-4 molecules,including novel soluble recombinant mutant CTLA-4-Ig fusion proteinsdescribed herein, which have a binding avidity for CD86 that is greaterthan the binding avidity of Belatacept (LEA29Y-Ig) for CD86. Theinvention also provides mutant CTLA-4 molecules, including novel solublerecombinant mutant CTLA-4-Ig fusion proteins described herein, whichhave a binding avidity for CD86 that is greater than the binding avidityof Belatacept for CD86. In another aspect, the invention provides mutantCTLA-4 molecules, including novel soluble recombinant mutant CTLA-4-Igfusion proteins described herein, which have a greater ability tosuppress one or more immune responses (e.g., T cell-dependent immuneresponses) than Belatacept. Mutant CTLA-4 molecules of the inventionhaving one or more improved properties compared to Belatacept areexpected to more potent than Belatacept and thus more effective, useful,and advantageous than Belatacept in treating diseases, disorders, orconditions in which immunosuppression is desirable, including thosediseases, disorders, or conditions for which the Belatacept fusionprotein has been shown to provide clinical benefit, such as renalallograft survival in non-human primates.

The safety, tolerability, pharmacokinetics, immunogenicity, and clinicalefficacy of a molecule of the invention, such as a mutant CTLA-4molecule of the invention, (e.g., mutant CTLA-4 ECD polypeptide orsoluble mutant CTLA-4-Ig fusion protein as described in detail below) ina subject having an immune disease or disorder (e.g., rheumatoidarthritis, multiple sclerosis, psoriasis, etc.) to whom a particulardose of the molecule is administered in a particular manner (e.g.,parenteral, intravenous, or subcutaneous administration) can bedetermined using methodologies comparable to those employed in clinicaltrials for Orencia® involving similar subjects. See, e.g., the worldwidewebsite addresses at bms.com and orencia.com. For example, the degree towhich a mutant CTLA-4 molecule of the invention (e.g., soluble mutantCTLA-4-Ig) is effective in reducing in subjects having rheumatoidarthritis (RA) the progression of joint damage, in alleviating the signsand symptoms of RA, including pain reduction, can be evaluated usingmethodologies similar to those employed in the Orencia® clinical trialsinvolving RA patients.

The safety, tolerability, pharmacokinetics, immunogenicity, and clinicalefficacy of a molecule of the invention (e.g., mutant CTLA-4 ECDpolypeptide or soluble mutant CTLA-4-Ig fusion protein as described indetail below) in a subject in which immunosuppression is desirable(e.g., a subject undergoing tissue, cell, organ, or grafttransplantation from a donor) and to whom a particular dose of themolecule is administered in a particular manner (e.g., parenteral,intravenous, or subcutaneous administration) can be determined usingmethodologies comparable to those employed in clinical trials forBelatacept involving similar subjects. See, e.g., the worldwide websiteaddress bms.com. For example, the degree to which a mutant CTLA-4molecule of the invention (e.g., soluble mutant CTLA-4-Ig) is effectivein reducing in kidney or renal transplant rejection in a recipientpatient undergoing kidney or renal transplantation can be evaluatedusing methodologies similar to those employed in the Belatacept clinicaltrial involving patients undergoing kidney or renal transplantation.

Molecules and methods of the invention and other aspects of theinvention are discussed in additional detail below.

Polypeptides of the Invention

The present invention provides novel polypeptides, collectively referredto as “polypeptides of the invention.” The term “polypeptides of theinvention” is intended to include variants and/or derivatives of thepolypeptide sequences disclosed herein. Polypeptides of the inventioninclude recombinant non-naturally occurring or mutant CTLA-4polypeptides that bind CD80 and/or CD86 and/or that inhibit or suppressimmune responses. Polypeptides of the invention include recombinantfusion proteins comprising a mutant CTLA-4 polypeptide of the invention,and include monomeric and dimer forms of such fusion proteins.Polypeptides of the invention include multimers comprising one or moremutant CTLA-4 polypeptides of the invention. The invention also includesconjugates comprising one or more mutant CTLA-4 polypeptides of theinvention. Some polypeptides of the invention are soluble polypeptides.For example, as described in more detail below, the invention includessoluble fusion proteins comprising a mutant CTLA-4 ECD polypeptidelinked to a different polypeptide (such as, e.g., an immunoglobulinpolypeptide, such as, e.g., an Ig Fc polypeptide) that enhancessolubility of the mutant CTLA-4 ECD polypeptide.

As discussed in greater detail below, in one aspect of the invention, avariety of mutagenesis and screening strategies were used to make andidentify novel polypeptides that bind CD80 and/or CD86. In particular,such strategies were used to make and identify novel polypeptides havingimproved abilities to bind CD80 and/or CD86, including novel mutantCTLA-4 polypeptides having improved binding affinities or avidities forCD80 and/or CD86. Polypeptides of the invention that bind CD80 and/orCD86 ligands expressed on antigen-presenting cells interfere with orblock the interaction of these ligands with the CD28 receptors expressedon T cells. As a result, the T cell costimulatory signal provided by theinteraction of the T cell surface receptor CD28 with the B7 molecules(i.e., CD80 and CD86) is inhibited or blocked. Such polypeptides arebelieved useful in the prophylactic and therapeutic treatment ofdiseases, disorders, and conditions in which modulation of the immunesystem (e.g., T cell responses) is of benefit.

Mutant CTLA-4 Polypeptides

In one aspect, the invention provides isolated or recombinantpolypeptides which each comprise a polypeptide sequence that has atleast 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%,92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%,98.5%, 99%, 99.5%, or 100% sequence identity to at least one polypeptidesequence selected from the group consisting of SEQ ID NOS:1-73 (e.g.,SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56,SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61,SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, and SEQ ID NO:73), wherein the polypeptide binds CD80and/or CD86 or a polypeptide fragment of CD80 and/or CD86 (or an ECD ofeither or both), and/or modulate or regulate an immune response. Somesuch polypeptides, such as each of those set forth in SEQ ID NOS:1-73,are described as secreted or mature mutant CTLA-4 ECD polypeptides. Themutant CTLA-4 ECD polypeptides set forth in each of SEQ ID NO:1-73 donot include a signal peptide; it has been cleaved during processing,thereby producing the mature or secreted polypeptide. A polypeptidefragment of CD80 may comprise, e.g., an extracellular domain polypeptideof a CD80 polypeptide, such as, e.g., human CD80 ECD polypeptide (“hCD80ECD”). A polypeptide fragment of CD86 may comprise, e.g., anextracellular domain polypeptide of a CD86 polypeptide, such as, e.g.,human CD86 ECD polypeptide (“hCD86 ECD”). Some such polypeptides bind amammalian CD80 and/or CD86 or polypeptide fragment thereof, such as,e.g., a mammalian CD80 or CD86 ECD. Some such polypeptides bind WT humanCD80 (“hCD80”) and/or WT human CD86 (“hCD86”) or a polypeptide fragmentthereof, such as, e.g., hCD80 ECD or hCD86 ECD. In some such methods, atleast one immune response is suppressed or inhibited.

Some such polypeptides have a binding affinity or avidity for hCD80 or afragment thereof (e.g., hCD80 ECD) that is at least about equal to orgreater than the binding affinity or avidity of the hCTLA-4 ECDpolypeptide for hCD80 or a fragment thereof, respectively. The predictedfull-length hCD80 polypeptide sequence, which includes a signal peptide,ECD, transmembrane domain, and cytoplasmic domain covalently linkedtogether in that order, is set forth in SEQ ID NO:195. The signalpeptide comprises amino acid residues 1-34, the ECD comprises amino acidresidues 35-242, the transmembrane domain comprises amino acid residues243-263, and the cytoplasmic domain comprises amino acid residues264-288 of SEQ ID NO:195. The polypeptide sequence of the hCD80 ECD isshown in SEQ ID NO:174. The nucleic acid sequence shown in SEQ ID NO:173encodes the WT human CD80 signal peptide (at N-terminus) and human CD80ECD.

Some such polypeptides have a binding affinity or avidity for hCD86 or afragment thereof (e.g., hCD86 ECD) that is at least about equal to orgreater than the binding affinity or avidity of the hCTLA-4 ECDpolypeptide for hCD86 or a fragment thereof (e.g., ECD), respectively.The predicted full-length hCD86 polypeptide sequence, which includes asignal peptide, ECD, transmembrane domain, and cytoplasmic domaincovalently linked together in that order, is set forth in SEQ ID NO:175,and an exemplary nucleic acid encoding the predicted full-length hCD86polypeptide sequence is shown in SEQ ID NO:176. The polypeptide sequenceof the hCD86 ECD is shown in SEQ ID NO:180. Some such polypeptides havea binding affinity or avidity for hCD86 that is at least about equal toor greater than the binding affinity or avidity of the LEA29Y ECDpolypeptide having the sequence set forth in SEQ ID NO:168 for hCD86.Exemplary polypeptides of the invention that have binding affinities orbinding avidities for hCD86 or hCD86 ECD that are at least equal to orgreater than those of hCTLA-4 ECD and LEA29Y ECD (also termed“A29YL104E” or “L104EA29Y” ECD) for hCD86 or hCD86 ECD, respectively,include, e.g., but are not limited to, those comprising a polypeptidesequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to the sequence of any of SEQ ID NOS:4, 10-12, 15, 17, 24, 26,28, 35, and 61. See, e.g., Table 5 in Example 4. The data presented inTable 5 reflect a monomeric interaction between a representativeCTLA-4-Ig and monomeric CD86 ECD. The term LEA29Y (or A29YL104E orL104EA29Y), if not otherwise indicated, refers to LEA29Y ECD (orA29YL104E or L104EA29Y ECD).

Some such polypeptides comprise a polypeptide sequence having an aminoacid length about equal to the amino acid length of the human CTLA-4extracellular domain. Such polypeptides may be described as mutantCTLA-4 ECD polypeptides. Some such mutant CTLA-4 ECD polypeptidescomprise a polypeptide sequence that is about 110 amino acids to about138 amino acid residues, about 112 to about 136 amino acid residues,about 114 to about 134 amino acid residues, about 116 to about 132 aminoacid residues, about 118 to about 130 amino acid residues, about 119 toabout 129 amino acid residues, about 120 to about 128 amino acidresidues, about 121 to about 127 amino acid residues, about 122 to about126 amino acid residues, about 123 to about 125 amino acid residues inlength. Some such mutant CTLA-4 ECD polypeptides comprise a sequencethat is 124 amino acid residues in length. Exemplary polypeptidesinclude, e.g., but are not limited to, a polypeptide comprising apolypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to at least one polypeptidesequence selected from the group consisting of SEQ ID NOS:1-73, whereinsuch polypeptide binds CD80 and/or CD86 (or an ECD of either or both).

Some such polypeptides described above, including, e.g., those isolatedor recombinant polypeptides which each comprising a polypeptide sequencehaving at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to atleast one polypeptide sequence selected from the group of SEQ IDNOS:1-73 and which bind CD80 and/or CD86 and/or an ECD thereof, have anability to modulate or regulate an immune response. One or more of avariety of immune responses may be modulated or regulated by suchpolypeptides of the invention, including, but not limited to, e.g., Tcell activation or proliferation, cytokine synthesis or production(e.g., production of TNF-α, IFN-γ, IL-2, etc.), induction of variousactivation markers (e.g., CD25, IL-2 receptor, etc.), synthesis orproduction of inflammatory molecules, inflammation, joint swelling,joint tenderness, pain, stiffness, serum levels of C-reactive protein,anti-collagen antibody production, and/or T cell-dependent antibodyresponse(s)). In some instances, such a polypeptide has a greaterability to suppress or inhibit at least one such immune response thanhCTLA-4 or hCTLA-4 ECD.

For example, some such polypeptides are capable of inhibiting T cellactivation or T cell proliferation in in vitro assays. Examples 4-9 setforth below, for example, demonstrate the ability of representativefusion proteins of the invention comprising a representative mutantCTLA-4 ECD polypeptide sequence, such as those described herein, toinhibit T cell proliferation in vitro. Some such polypeptides arecapable of inhibiting or suppressing an immune response in a subject invivo, such as through the administration of a therapeutically orprophylactically effective amount of at least one such polypeptide to asubject needing immunosuppressive therapy. Some such polypeptides areexpected to be useful in a variety of applications, including, e.g., butnot limited to, prophylactic and/or therapeutic methods for treatingimmune system diseases, disorders, and conditions in whichimmunomodulation is desirable, as discussed in greater detail infra.Such polypeptides are expected to be useful in prophylactic and/ortherapeutic methods for inhibiting or suppressing an immune response ina subject (e.g., in the in vivo treatment of immune system diseases ordisorders of mammals, such as e.g., humans, in which immunoinhibition orimmunosuppression is desirable), methods for inhibiting rejection of atissue or organ transplant from a donor by a recipient (e.g., by amammal, such as, e.g., a human), and other methods described elsewhereherein.

Additionally or alternatively, some such polypeptides have an ability tosuppress or inhibit an immune response that is at least about equal toor greater than the ability of hCTLA-4 or hCTLA-4 ECD to suppress orinhibit one or more types of immune responses. For example, some suchpolypeptides have an ability to inhibit T cell activation orproliferation in in vitro and/or in vivo assays and/or applications,such as those described above and in greater detail below, which is atleast about equal to or greater than the ability of hCTLA-4 or hCTLA-4ECD to inhibit T cell activation or proliferation in such applications.Additionally, some such polypeptides have an ability to inhibit orsuppress an immune response (e.g., T cell activation or proliferation,cytokine production, T cell-dependent antibody response) that is greaterthan the ability of a LEA29Y polypeptide—a specific mutant CTLA-4 ECDcomprising the polypeptide sequence shown in SEQ ID NO:168—to inhibit orsuppress an immune response. Examples 4-9 set forth below, for example,compare the ability of representative fusion proteins of the inventioncomprising a mutant CTLA-4 ECD polypeptide sequence of the invention toinhibit T cell proliferation in vitro relative to the ability of afusion protein comprising the hCTLA-4 ECD or LEA29Y polypeptide toinhibit T cell proliferation in vitro. Such molecules are expected to beof beneficial use in a variety of therapeutic applications, includingtreatment of autoimmune diseases and disorders, and prophylactic andtherapeutic methods for inhibiting rejection of organ, cell, or tissuegraft transplantation.

Some such polypeptides may differ from one another by, e.g., an aminoacid deletion, addition and/or substitution. An amino acid substitutionmay be a conservative or non-conservative substitution. See, e.g., thesection entitled “Sequence Variation.”

In another aspect, the invention also provides isolated or recombinantpolypeptides which each comprise a polypeptide sequence having at least95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least onepolypeptide sequence selected from the group consisting of SEQ IDNOS:1-73, wherein the polypeptide binds a monomeric hCD80 or monomerichCD86 or an ECD of either or both. Some such polypeptides have (1) abinding affinity for monomeric hCD86 that is about equal to or greaterthan the binding affinity of monomeric hCTLA-4 or an LEA29Y polypeptidefor monomeric hCD86 or an ECD thereof, and (2) a binding affinity formonomeric hCD80 that is about equal to or greater than the bindingaffinity of monomeric hCTLA-4 for monomeric hCD80. The LEA29Ypolypeptide comprises the polypeptide sequence of SEQ ID NO:168.Further, some such polypeptides have a greater ability to suppress oneor more immune responses described herein (e.g., T cellactivation/proliferation, cytokine synthesis/production, induction ofactivation markers, production of inflammatory molecules, inflammation,anti-collagen Ab production, T cell-dependent Ab response) thanmonomeric hCTLA-4, monomeric hCTLA-4 ECD, or LEA29Y polypeptide.

The invention also provides isolated or recombinant polypeptides whicheach comprise a polypeptide sequence having at least 95%, 96%, 97%, 98%,99%, or 100% sequence identity to at least one polypeptide sequenceselected from the group consisting of SEQ ID NOS:1-73, wherein thepolypeptide has a binding affinity for a hCD86 ECD or hCD80 ECD that isabout equal to or greater than the binding affinity of a hCTLA-4 ECD forthe hCD86 ECD or hCD80 ECD, respectively. Some such polypeptides have abinding affinity for the hCD86 ECD that is greater than the bindingaffinity of the hCTLA-4 ECD (SEQ ID NO:159) or the LEA29Y polypeptide(SEQ ID NO:168) for the hCD86 ECD. Some such polypeptides have a bindingaffinity for the hCD80 ECD that is greater than the binding affinity ofthe hCTLA-4 ECD for the hCD80 ECD. Some such polypeptides have anability to suppress an immune response, in some instances, a greaterability to suppress one or more immune responses, including thosedescribed above and throughout, than the hCTLA-4 ECD or the LEA29Ypolypeptide.

In another aspect, the invention provides an isolated or recombinantCTLA-4 polypeptide variant comprising a polypeptide sequence which (a)which differs from a polypeptide sequence of human CTLA-4 ECD shown inSEQ ID NO:159 in no more than 15 amino acid residues, no more than 14amino acid residues, no more than 13 amino acid residues, no more than12 amino acid residues, no more than 11 amino acid residues, no morethan 10 amino acid residues, no more than 9 amino acid residues, no morethan 8 amino acid residues, no more than 7 amino acid residues, no morethan 6 amino acid residues, no more than 5 amino acid residues (e.g., nomore than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acidresidues), and (b) wherein the amino acid residue at position 24, 30,32, 39, 41, 50, 54, 55, 56, 64, 65, 70, 85, 104 or 106 of the hCTLA-4ECD polypeptide sequence (SEQ ID NO:159) is substituted with a differentamino acid residue in the CTLA-4 polypeptide variant sequence, whereinamino acid residue positions of the CTLA-4 polypeptide variant arenumbered according to SEQ ID NO:159, and wherein the CTLA-4 polypeptidevariant has an ability to bind CD80 or CD86 or an extracellular domainor fragment of either, and/or has an ability to suppress or inhibit animmune response. Some such variants comprise one or more (e.g., one,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen or fifteen) amino acid substitutions selected fromthe group consisting of A24E, A24S, S25A, G27H, K28N, T30N, T30D, T30A,V32I, Q39K, Q39E, D41G, D41N, D41S, A50M, A50G, M54K, M54E, M54V, G55E,G55K, N56D, D63K, S64P, I65S, I65F, I65T, S70F, M85A, M85V, M85A, L104E,L104D, and I106M, I106F, and I106L.

In another aspect, the invention provides an isolated or recombinantmutant CTLA-4 polypeptide comprising a polypeptide sequence which (a)differs from the polypeptide sequence of the extracellular domain ofhuman CTLA-4 shown in SEQ ID NO:159 in no more than 12 amino acidresidues (e.g., no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 aminoacid residues), and (b) comprises two, three, four, five, six, seven,eight, nine, ten, eleven, or 12 amino acid substitutions selected fromthe group consisting of amino acid positions corresponding to amino acidpositions 24, 30, 32, 50, 54, 55, 56, 64, 65, 70, and 104 of SEQ IDNO:159, wherein amino acid positions of the mutant CTLA-4 polypeptideare numbered according to SEQ ID NO:159, and wherein the mutant CTLA-4polypeptide has an ability to bind CD80 or CD86 or an extracellulardomain or fragment of either, and/or has an ability to suppress orinhibit an immune response. Exemplary amino acid substitutions in thesepositions include, but are not limited to, conservative amino acidsubstitutions of the amino acid residues present in WT hCTLA-4 ECDand/or A24S/E (that is, A24S or A24E), T30N/D/A (that is, T30N or T30Dor T30A), V32I/L/M/V, A50M/G, M54E/V/K, G55E/K/R, N56D/A, S64P/M/C,I65S/T/F/V, S70F/Y/W, L104E/M, or any combination of substitutionsthereof.

In another aspect, the invention provides isolated or recombinantpolypeptides (e.g., mutant CTLA-4 ECD polypeptides) which each comprisea polypeptide sequence (a) which differs from a polypeptide sequenceselected from the group consisting of SEQ ID NOS:1-73 in no more than14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue(s),and (b) wherein the amino acid residue in the polypeptide sequence atamino acid residue position 41, 50, 54, 55, 56, 64, 65, 70, or 85 isidentical to the amino acid residue at the corresponding position ofsaid selected polypeptide sequence (e.g., a polypeptide selected fromSEQ ID NOS:1-73), wherein the polypeptide binds CD80 and/or CD86 and/oran extracellular domain of either or both, and/or inhibits an immuneresponse(s). In some instances, the polypeptide differs from theselected polypeptide (e.g., selected from SEQ ID NOS:1-73) in no morethan 10, 9, 8, 7, or 6 amino acid residues (e.g., no more than 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 amino acid residues), but the amino acidoccupying one or more of amino acid residue positions 41, 50, 54, 55,56, 64, 65, 70, and 85 is identical to the amino acid residue includedat that position in the selected polypeptide sequence (e.g., oneselected from SEQ NOS: 1-73) and is not deleted or substituted withanother amino acid. Some such polypeptides comprise a polypeptidesequence which differs from the selected polypeptide sequence in no morethan 10, 9, 8, 7, or 6 amino acid residues (e.g., no more than 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 amino acid residues) and which includes aminoacid residues at one or more of amino acid residue positions 24, 30, 32,41, 50, 54, 55, 56, 64, 65, 70, 85, 104 and 106 that are identical tothe amino acid residues at the corresponding positions in the selectedpolypeptide sequence. Such polypeptides can differ from the selectedpolypeptide sequence by amino acid deletion(s), addition(s), and/oramino acid substitution(s) at a position(s) that are not specified ashaving an amino acid identical to that of the selected sequence. Suchpolypeptides having a binding affinity or avidity for hCD86 or afragment thereof (e.g., hCD86 ECD) that is at least about equal to orgreater than the binding affinity or avidity of the hCTLA-4 ECD orLEA29Y polypeptide for hCD86 or a fragment thereof (e.g., ECD),respectively, are included. Some such polypeptides have a bindingaffinity or avidity for hCD80 or a fragment thereof (e.g., hCD80 ECD)that is at least about equal to or greater than the binding affinity oravidity of the hCTLA-4 ECD polypeptide for hCD80 or a fragment thereof,respectively. Some such polypeptides comprise a polypeptide sequencehaving a length about equal to the amino acid length of the hCTLA-4 ECD,e.g., 118-130, 119-129, 120-128, 121-127, 122-126, 123-125, or 124 aminoacid residues in length.

Some such polypeptides are capable of suppressing one or more of avariety of immune responses, including, e.g., T cell activation, T cellproliferation, cytokine synthesis or production (e.g., production ofTNF-α, IFN-γ, IL-2), induction of activation markers (e.g., CD25, IL-2receptor), inflammation, production of inflammatory molecules,anti-collagen Ab production, and/or T cell-dependent Ab response(s)).Some such polypeptides have a greater ability to inhibit one or moresuch immune responses than hCTLA-4, hCTLA-4 ECD polypeptide, or LEA29Ypolypeptide. For example, some such polypeptides are capable ofinhibiting T cell activation or T cell proliferation in in vitro assays.Examples 4-9, e.g., compare the ability of representative fusionproteins of the invention comprising a mutant CTLA-4 ECD polypeptidesequence of the invention to inhibit T cell proliferation in vitrorelative to the ability of a fusion protein comprising the hCTLA-4 ECDor LEA29Y polypeptide to do so. Some such polypeptides are capable ofinhibiting or suppressing an immune response in a subject in vivo, suchas through the administration of a therapeutically or prophylacticallyeffective amount of at least one such polypeptide to a subject needingimmunosuppressive therapy. Such polypeptides are expected to be usefulin a variety of applications, including, e.g., but not limited to,prophylactic and/or therapeutic methods for treating immune systemdiseases, disorders, and conditions in which suppression of an immuneresponse is desirable, including, e.g., prophylactic and/or therapeuticmethods for treating autoimmune diseases and disorders, methods forinhibiting rejection of a tissue or organ transplant from a donor by arecipient (e.g., by a mammal, such as, e.g., a human), and other methodsdescribed elsewhere herein.

In another aspect, the invention provides isolated or recombinantpolypeptides (e.g., mutant CTLA-4 ECD polypeptides) which each comprisea polypeptide sequence which (a) differs from the polypeptide sequenceof the extracellular domain of human CTLA-4 shown in SEQ ID NO:159 in nomore than 10 amino acid residues, no more than 9 amino acid residues, nomore than 8 amino acid residues, no more than 7, amino acid residues, nomore than 6 amino acid residues, no more than 5 amino acid residues, nomore than 4 amino acid residues, no more than 3 amino acid residues, nomore than 2 amino acid residues, or no more than 1 amino acid residue(e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidresidues), and (b) comprises at least one amino acid substitution at anamino acid residue position corresponding to position 50, 54, 55, 56,64, 65, 70, or 85 relative to the polypeptide sequence of SEQ ID NO:159,wherein the polypeptide binds hCD80 and/or hCD86 and/or an ECD of eitheror both, and/or inhibits an immune response (e.g., T cell activation orproliferation, cytokine synthesis or production (e.g., production ofTNF-α, IFN-γ, IL-2), induction of activation markers (e.g., CD25, IL-2receptor), inflammation, production of inflammatory molecules,anti-collagen Ab production, and/or T cell-dependent Ab response), suchas in in vitro and/or in vivo methods and/or assays as described ingreater detail below. Some such polypeptides comprise a polypeptidesequence that is 124 amino acid residues in length. Some suchpolypeptides comprise 2, 3, 4, 5, or 6 amino acid substitutions atpositions relative to the polypeptide sequence set forth in SEQ IDNO:159 selected from the group consisting of amino acid residue position50, 54, 55, 56, 64, 65, 70, and 85. Some such polypeptides furthercomprise an amino acid substitution at an amino acid residue positioncorresponding to position 104 and/or 30 relative to SEQ ID NO:159. Somesuch polypeptides comprise at least one amino acid substitution relativeto SEQ ID NO:159 at position 70 (optionally S70F), position 64(optionally S64P), position 50 (optionally A50M/G, e.g., A50M, A50G),position 54 (optionally M54K/V, e.g., M54K), position 65 (optionallyI65S), position 56 (optionally N56D), position 55 (optionally G55E/K,e.g., G55E, G55K), position 85 (optionally M85A), and/or position 24(optionally A24E/S, e.g., A24E). Any such polypeptide may furthercomprise an amino acid substitution relative to SEQ ID NO:159 atposition 104 (optionally L104E/D, e.g., L104E), position 30 (optionallyT30N/D/A, e.g., T30N, T30D, or T30A), and/or position 32 (optionallyV32I). In some instances, the polypeptide comprises one or moresubstitutions at amino acid positions relative to SEQ ID NO:159 selectedfrom the group consisting of A50M, M54K, G55E, N56D, S64P, I65S, andS70F. Some such polypeptides exhibit a binding affinity for CD86 (e.g.,hCD86) or CD86 ECD (e.g., hCD86 ECD) that is about equal to or greaterthan the binding affinity of a monomeric hCTLA-4 ECD for CD86 or CD86ECD, respectively. Some such polypeptides exhibit a binding affinity forCD80 (e.g., hCD80) or CD80 ECD (e.g., hCD80 ECD) that is greater thanthe binding affinity of a monomeric hCTLA-4 ECD for CD80 or CD80 ECD,respectively.

Some such polypeptides have an ability to suppress or inhibit one ormore immune responses (e.g., T cell activation or proliferation,cytokine production, etc.), such as in vitro and/or in vivo. Some suchpolypeptides inhibit one or more such immune responses to a greaterdegree than hCTLA-4, hCTLA-4 ECD, or LEA29Y polypeptide. Suchpolypeptides are expected to be of beneficial use in a variety oftherapeutic applications, including prophylactic and/or therapeuticmethods for treating autoimmune diseases and disorders, or prophylacticand/or therapeutic methods for inhibiting organ, cell, or tissue grafttransplantation rejection.

In another aspect, the invention provides isolated or recombinantpolypeptides (e.g., mutant CTLA-4 ECD polypeptides) which each comprisea polypeptide sequence which (a) differs from the polypeptide sequenceof the extracellular domain of human CTLA-4 shown in SEQ ID NO:159 in nomore than 6 amino acid residues (e.g., no more than 1, 2, 3, 4, 5, or 6amino acid residues), and (b) comprises one or more substitutions atamino acid positions relative to SEQ ID NO:159 selected from the groupconsisting of A50M, M54K, G55E, N56D, S64P, I65S, and S70F, wherein thepolypeptide binds hCD80 and/or hCD86 or an extracellular domain ofeither or both, and/or inhibits an immune response (e.g., T cellactivation or proliferation, cytokine synthesis or production, inductionof activation markers, production of inflammatory molecules,inflammation, joint swelling or tenderness, pain, stiffness, serumlevels of C-reactive protein, anti-collagen Ab production, and/or Tcell-dependent Ab response, etc.) in in vitro assays and/or in vivomethods. Such polypeptides are expected to be useful in treating asubject suffering from a disease, disorder, or condition in whichimmunosuppressive therapy would be of benefit, including, e.g.,therapeutic and prophylactic methods for treating autoimmune diseasesand disorders, and prophylactic and therapeutic methods for inhibitingorgan, cell, or tissue graft transplantation.

The invention also includes an isolated or recombinant polypeptide whichcomprises a polypeptide sequence comprising (i) at least 95%, 96%, 97%,98%, 99%, or 100% sequence identity to any polypeptide sequence selectedfrom the group consisting of SEQ ID NOS:1-73 and (ii) a phenylalanineresidue at an amino acid position corresponding to position 70 of saidpolypeptide sequence selected from the group consisting of SEQ IDNO:1-73, wherein the polypeptide binds hCD80 and/or hCD86 or anextracellular domain thereof and/or inhibits an immune response (e.g., Tcell activation or proliferation, cytokine synthesis or production,induction of activation markers, production of inflammatory molecules,inflammation, joint or tenderness, pain, stiffness, serum levels ofC-reactive protein, anti-collagen Ab production, and/or T cell-dependentAb response, etc.) in in vitro assays and/or in vivo methods. Some suchpolypeptides comprise one or more of the following relative to theselected sequence: a glutamic acid residue at an amino acid positioncorresponding to position 24; an asparagine residue at an amino acidposition corresponding to position 30; an isoleucine residue at an aminoacid position corresponding to position 32; a methionine residue at anamino acid position corresponding to position 50; a lysine residue at anamino acid position corresponding to position 54; a glutamic acidresidue at an amino acid position corresponding to position 55; anaspartic acid residue at an amino acid position corresponding toposition 56; a proline residue at an amino acid position correspondingto position 64; a serine residue at an amino acid position correspondingto position 65; and a glutamic acid residue at an amino acid positioncorresponding to position 104. Such polypeptides are expected to be ofbeneficial use in a variety of applications, including methods fortreating autoimmune diseases and disorders, and methods for inhibitingorgan, cell or tissue graft transplantation.

For example, in one non-limiting aspect, the invention includes anisolated or recombinant polypeptide (e.g., mutant CTLA-4 ECD) whichcomprises a polypeptide sequence comprising (i) at least 95%, 96%, 97%,98%, 99%, or 100% identity to the polypeptide sequence of SEQ ID NO:24and (ii) a phenylalanine residue at an amino acid position correspondingto position 70 of the polypeptide sequence of SEQ ID NO:24, wherein thepolypeptide binds hCD80 and/or hCD86 and/or an ECD of either or both,and/or inhibits an immune response in vitro and/or in vivo. Thepolypeptide may comprise at least one of the following relative to SEQID NO:24: a glutamic acid residue at position 24; an asparagine residueat position 30; an isoleucine residue at position 32; a methionineresidue at position 50; a lysine residue at position 54; a glutamic acidresidue at position 55; an aspartic acid residue at position 56; aproline residue at position 64; a serine residue at position 65; and aglutamic acid residue at position 104.

The invention also includes an isolated or recombinant polypeptide(e.g., mutant CTLA-4 ECD polypeptide) that binds hCD80 and/or hCD86(and/or an ECD of either or both) and/or inhibits an immune response (asdescribed above), e.g., in vitro and/or in vivo, wherein the polypeptidecomprises a polypeptide sequence which (a) differs from the polypeptidesequence of human CTLA-4 ECD polypeptide shown in SEQ ID NO:159 in nomore than 6 amino acid residues, and (b) comprises at least one aminoacid substitution, wherein said at least amino acid substitutioncomprises S70F, wherein amino acid residue positions are numberedaccording to SEQ ID NO:159. The polypeptide may further comprise atleast one amino acid substitution selected from the group consisting ofA24E, T30N, V32I, D41G, A50M, M54K, G55E, N56D, S64P, I65S, M85A, L104E,and I106F. Such polypeptides believed useful in a variety ofapplications, including methods for treating autoimmune diseases anddisorders, and methods for inhibiting organ, cell, tissue, or grafttransplantation.

The invention also includes an isolated or recombinant polypeptidecomprising a polypeptide sequence which (a) differs from the polypeptidesequence shown in SEQ ID NO:31 in no more than 6 amino acid residues,and (b) comprises at least one of the following: a methionine residue ata position corresponding to position 50 of SEQ ID NO:31, a lysineresidue at a position corresponding to position 54 of SEQ ID NO:31, aglutamic acid residue at a position corresponding to position 55 of SEQID NO:31, a proline residue at a position corresponding to position 64of SEQ ID NO:31, a serine residue at a position corresponding toposition 65 of SEQ ID NO:31, a phenylalanine residue at a positioncorresponding to position 70 of SEQ ID NO:31, wherein amino acid residuepositions are numbered according to SEQ ID NO:31, and the polypeptidebinds CD80 and/or CD86 and/or an ECD of either or both, and/or inhibitsan immune response as described above in vitro and/or in vivo. Thepolypeptide may comprise a glutamic acid residue at a positioncorresponding to position 104, an asparagine acid residue at a positioncorresponding to position 30, and/or an isoleucine residue at a positioncorresponding to position 32 of SEQ ID NO:31. Such polypeptides believeduseful in a variety of applications, including methods for treatingrheumatic diseases and disorders, and methods for inhibiting organ, cellor tissue graft transplantation.

In another aspect, the invention provides an isolated or recombinantpolypeptide which comprises a polypeptide sequence comprising (i) atleast 95%, 96%, 97%, 98%, or 99% sequence identity to a polypeptidesequence selected from the group consisting of SEQ ID NOS:36-46 and 55,and (ii) a glutamic acid residue at an amino acid position 55 of saidselected polypeptide sequence, wherein the polypeptide binds CD80 and/orCD86 or an extracellular domain of either or both and/or suppresses animmune response. Immune responses that can be suppressed include, e.g.,T cell activation or proliferation, cytokine synthesis or production(e.g., production of TNF-α, IFN-γ, IL-2), induction of activationmarkers (e.g., CD25, IL-2 receptor), inflammation, production ofinflammatory molecules, anti-collagen Ab production, and/or Tcell-dependent Ab response). Such polypeptide sequence may furthercomprise a phenylalanine residue at amino acid position 70. Suchpolypeptide sequence may further comprise a proline residue at position64 and/or an asparagine residue at position 30. Such polypeptidesequence may further comprise a methionine residue at position 50 and/ora lysine residue at position 54.

In another aspect, the invention provides an isolated or recombinantpolypeptide which comprises a polypeptide sequence comprising (i) atleast 95%, 96%, 97%, 98%, or 99% sequence identity to a polypeptidesequence selected from the group consisting of SEQ ID NOS:28, 30, 36-46,55-57, and 65-73, and (ii) a glutamic acid residue at an amino acidposition 55 of said selected polypeptide sequence, wherein thepolypeptide binds CD80 and/or CD86 or an extracellular domain of eitheror both and/or suppresses an immune response, such as T cell activationor proliferation, cytokine synthesis or production (e.g., production ofTNF-α, IFN-γ, IL-2), induction of activation markers (e.g., CD25, IL-2receptor), inflammation, production of inflammatory molecules,anti-collagen Ab production, and/or T cell-dependent Ab response. Suchpolypeptide sequence may further comprise a phenylalanine residue atamino acid position 70. Such polypeptide sequence may further comprise aproline residue at position 64 and/or an asparagine residue at position30. Such polypeptide sequence may further comprise a methionine residueat position 50 and/or a lysine residue at position 54.

Any polypeptide of the invention described above may further include apeptide that facilitates secretion of said polypeptide. Thus, in oneaspect, the invention provides an isolated or recombinant polypeptidecomprising (a) any polypeptide as described above (e.g., a mutant CTLA-4ECD described above), and (b) a peptide that facilitates secretion ofthe expressed polypeptide from a host cell. The peptide is optionally asignal peptide. The C-terminus of the signal peptide is typicallycovalently linked to the N-terminus of the polypeptide. The signalpeptide may comprise an amino acid sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO:182 or SEQ ID NO:216. The signalpeptide may comprise an amino acid sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an aminoacid sequence comprising amino acid residues 1-35, 1-36, or 1-37 of SEQID NO:160.

Any polypeptide of the invention described above may further comprise atransmembrane domain and/or cytoplasmic domain. Thus, in one aspect, theinvention provides an isolated or recombinant polypeptide comprising (a)any polypeptide of the invention described above (e.g., a mutant CTLA-4ECD described above), and (b) a transmembrane domain. Such protein mayoptionally further comprise a signal peptide as described above, whereinthe C-terminus of the signal peptide is covalently linked to theN-terminus of the polypeptide of the invention. The C-terminus of thesignal peptide is typically covalently linked to the N-terminus of thetransmembrane domain. The C-terminus of the transmembrane domain istypically covalently linked to the N-terminus of the cytoplasmic domain.In some instances, the transmembrane domain comprises an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identity to an amino acid sequence comprising amino acidresidues 162-182 of SEQ ID NO:160. In some instances, the cytoplasmicdomain comprises an amino acid sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acidsequence comprising amino acid residues 183-223 of SEQ ID NO:160. Any ofthe above-described polypeptides may comprise one or more of the aminoacid residues that are glycosylated or pegylated.

The invention also includes isolated or recombinant polypeptidemultimers comprising two or more polypeptides, wherein at least one ofthe polypeptides of the multimer is a mutant CTLA-4 polypeptide of theinvention as described herein (e.g., a mutant CTLA-4 ECD or mutantCTLA-4-Ig). Such a multimer comprises at least one polypeptide of theinvention and may further comprise at least one additional polypeptidethat need not be a polypeptide of the invention. For example, themultimer may comprise at least one polypeptide of the invention and atleast one other polypeptide which may be, e.g., a wild-type polypeptide(e.g., hCTLA-4 ECD or hCTLA-4-Ig) and/or at least other mutantpolypeptide (such as a mutant polypeptide that is not a polypeptide ofthe invention). Some or all of the polypeptides in the multimer (ormultimeric polypeptide) may be identical to one another, or, in someinstances, all polypeptides in the multimer may be different from oneanother. In some instances, the polypeptide multimer is a dimercomprising two polypeptides of the invention, which optionally may beidentical polypeptides (i.e., homodimer) or different polypeptides(i.e., heterodimer). In some instances, the polypeptide multimer is atetramer comprising four polypeptides of the invention. The tetramer cancomprise four identical polypeptides (i.e., homotetramer) or anycombination of four polypeptides of the invention such that at least onepolypeptide is not identical to the other three polypeptides (i.e.,heterotetramer). The invention also includes a tetramer comprising fouridentical WT CTLA-4 ECD polypeptides (e.g., hCTLA-4 ECD) or fouridentical WT CTLA-4-Ig (e.g., hCTLA-4-Ig). In some instances, themultimer is capable of binding CD80 and/or CD86 (or an ECD of either orboth) and/or suppressing or inhibiting an immune response in in vitroand/or in vivo methods (e.g., T cell proliferation or activation,cytokine production, etc.). Some such multimers have a greater abilityto suppress or inhibit an immune response in vitro and/or in vivo thanhCTLA-4 or hCTLA-4-Ig (e.g., hCTLA-4-IgG2 or Orencia® protein). Thepolypeptides of the multimers may be linked together, such as bycovalent linkages, such as via disulfide bonds between one or morecysteine residues in the one or more polypeptides.

Some such tetramers of the invention comprise a structure schematicallysimilar to that of an antibody, but in which the variable domains of theantibody are each replaced with any mutant CTLA-4 ECD polypeptide of theinvention described herein. The heavy chain of an antibody comprises aheavy chain variable domain (V_(H)) fused to an immunoglobulin (Ig) CH1domain (e.g., IgG2 CH1), which is fused to a hinge. The hinge is fusedto an Ig CH2 domain (e.g., IgG2 CH2), which is fused to an Ig CH3 domain(e.g., IgG2 CH3). The light chain of an antibody comprises a light chainvariable domain (V_(L)) fused to an Ig C kappa (C_(κ)) or C lambda(C_(λ)) domain. Two heavy chains and two light chains are covalentlylinked together by one or more disulfide bonds formed via cysteineresidues in the heavy and light chains. The invention includes a mutantCTLA-4 tetramer in which each of the variable domains of the heavy andlight chains is replaced with a mutant CTLA-4 ECD polypeptide of theinvention. Thus, such tetramer comprises two light chains and two heavychains. Each light chain comprises a mutant CTLA-4 ECD polypeptide fusedto an Ig C_(κ) or C_(λ) domain. Each heavy chain comprises a mutantCTLA-4 ECD fused to an Ig CH1 domain (e.g., IgG2 CH1), which is fused toa hinge. The hinge is fused to an Ig CH2 domain (e.g., IgG2 CH2), whichis fused to an Ig CH3 domain (e.g., IgG2 CH3). The two heavy chains andtwo light chains are covalently linked together by one or more disulfidebonds formed via cysteine residues in the heavy and light chains. Suchtetramer may be described as a CTLA-4-Ig tetramer. Methods forconstructing such CTLA-4-Ig tetramer are known and would be understoodby those of ordinary skill in the art. A tetrameric CD4-Ig construct,which comprises a CD4 polypeptide and which neutralizes primary HIV type1 isolates, is described in Allaway, G. P. et al., AIDS Res. Hum.Retroviruses 11(5):533-9 (1995). The tetramer can comprise fouridentical four mutant CTLA-4 ECD polypeptides or any combination of fourmutant CTLA-4 ECD polypeptides of the invention such that at least onemutant CTLA-4 ECD is not identical to the other three mutant CTLA-4 ECDpolypeptides. Some such tetramers are capable of binding CD80 and/CD86with a higher binding avidity than hCTLA-4 (or hCTLA-4-Ig). Some suchtetramers are capable of suppressing or inhibiting an immune response;in some instances, such a tetramer has a greater ability to suppress orinhibit an immune response in in vitro assays or in vivo applications(e.g., T cell proliferation or activation, cytokine production, etc)than hCTLA-4 or hCTLA-4-Ig (e.g., hCTLA-4-IgG2 or Orencia®). Multimersof the invention are expected to be of beneficial use in a variety ofapplications, including methods for treating autoimmune diseases anddisorders, and methods for inhibiting organ, cell, or tissue grafttransplantation.

The invention also includes an isolated or recombinant conjugatemultimers comprising two or more conjugates, wherein at least one of theconjugates is a conjugate of the invention which comprises a mutantCTLA-4 polypeptide of the invention (e.g., a mutant CTLA-4 ECD or mutantCTLA-4-Ig). Some or all of the conjugates in the multimer may beidentical to one another, or all conjugates in the multimer may bedifferent from one another. In some instances, the conjugate multimer isa dimer comprising two conjugates or a tetramer comprising fourconjugates of the invention. Some such conjugate multimers are capableof binding CD80 and/CD86 (or an ECD of either or both) and/orsuppressing or inhibiting an immune response in vitro and/or in vivo.Conjugate molecules in multimers may be linked together, such as bycovalent linkages, such as via disulfide bonds between one or morecysteine residues in the one or more conjugates.

The invention includes an isolated or recombinant polypeptide dimercomprising any two polypeptides of the invention described above (e.g.,mutant CTLA-4 ECD described above), wherein the dimer has a bindingavidity for human CD86 or an extracellular domain thereof that is aboutequal to or greater than the binding avidity of a dimer comprising twohuman CTLA-4 extracellular domains for human CD86 or an extracellulardomain thereof, respectively.

The invention includes isolated or recombinant polypeptide dimerscomprising two polypeptides of the invention described above (e.g.,mutant CTLA-4 ECD described above), wherein the dimer has a bindingavidity for hCD80 or an ECD thereof that is about equal to or greaterthan the binding avidity of a dimer comprising two hCTLA-4 ECDpolypeptides (SEQ ID NO:159) for hCD80 or an ECD thereof, respectively.For example, in a non-limiting aspect, the dimer may comprise twopolypeptides, wherein each polypeptide comprises a polypeptide sequencehaving at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequenceselected from the group consisting of SEQ ID NOS:1-73, and eachpolypeptide binds hCD80 and/or hCD86 and/or inhibits an immune response.In another non-limiting aspect, the dimer may comprise two polypeptides,wherein each polypeptide differs from the polypeptide sequence of thehCTLA-4 ECD (SEQ ID NO:159) in no more than 6 amino acid residues andcomprises at least one substitution at an amino acid position relativeto SEQ ID NO:159 selected from the group consisting of A50M, M54K, G55E,N56D, S64P, I65S, and S70F; and which polypeptide optionally furthercomprises the substitution L104E, and each polypeptide binds hCD80and/or hCD86 and/or inhibits an immune response.

In some instances, the dimer has a binding avidity for hCD80 or an ECDthereof that is about equal to or greater than the binding avidity of adimer comprising two hCTLA-4 ECD polypeptides for hCD80 or an ECDthereof, respectively. In some instances, the dimer has a bindingavidity for hCD86 or an ECD thereof that is greater than the bindingavidity of a dimer comprising two LEA29Y polypeptides for hCD86 or anECD thereof, respectively, wherein each LEA29Y polypeptide comprises thepolypeptide sequence shown in SEQ ID NO:168. In some instances, thedimer dissociates from binding hCD86 or an ECD thereof at a rate that isless than the rate at which a dimer comprising two hCTLA-4 ECDpolypeptides dissociates from binding hCD86 or an ECD thereof,respectively. In some instances, the dimer dissociates from bindinghCD86 or an ECD thereof at a rate that is less than the rate at which adimer comprising two LEA29Y polypeptides dissociates from binding hCD86or an ECD thereof, respectively, wherein each LEA29Y polypeptidecomprises the polypeptide sequence shown in SEQ ID NO:168.

In some instances, the dimer associates with hCD86 or an ECD thereof ata rate that is greater than the rate at which a dimer comprising twohCTLA-4 ECD polypeptides associates with hCD86 or an ECD thereof,respectively. In some instances, the dimer associates with hCD86 or anECD thereof at a rate that is greater than the rate at which a dimercomprising two LEA29Y polypeptides associates with hCD86 or an ECDthereof, respectively, wherein each LEA29Y polypeptide comprises thepolypeptide sequence shown in SEQ ID NO:168.

In some instances, such dimer comprising a mutant CTLA-4 ECD has agreater ability to suppress an immune response (e.g., T cell activationor proliferation, cytokine production, etc.) than a dimer comprising twohuman CTLA-4 extracellular domains or two LEA29Y polypeptides.

Some such dimers have a CD86 equilibrium dissociation constant (K_(D))that is less than the CD86 equilibrium dissociation constant (K_(D)) ofa dimer comprising two hCTLA-4 ECD polypeptides or two LEA29Ypolypeptides. Some such dimers have a CD86 equilibrium dissociationconstant (K_(D)) that is less than the CD86 equilibrium dissociationconstant (K_(D)) of a dimer comprising two LEA29Y polypeptides, eachLEA29Y polypeptide comprising the polypeptide sequence set forth in SEQID NO:168.

Some such multimers comprising at least two polypeptides of theinvention (e.g., a dimer comprising two mutant CTLA-4 ECD polypeptidesof the invention) have an enhanced ability to suppress an immuneresponse compared to a multimer of full-length hCTLA-4 of the samevalency (i.e., a multimer comprising the same number of full-lengthCTLA-4 polypeptides). Some such multimers comprising at least twopolypeptides of the invention have an enhanced ability to suppress animmune response compared to a multimer of hCTLA-4 ECCD of the samevalency (i.e., a multimer comprising the same number of hCTLA-4 ECDpolypeptides).

Any polypeptide of the invention described above may further comprise anadditional polypeptide sequence that enhances solubility, such as animmunoglobulin (Ig) polypeptide sequence, thereby forming, e.g., asoluble fusion protein, as discussed in greater detail infra. Eachpolypeptide of a polypeptide multimer may further comprise an additionalpolypeptide sequence that enhances solubility, such as an Ig polypeptidesequence, thereby forming, e.g., a soluble fusion protein. Thus, forexample, each polypeptide of a dimer comprising two or more polypeptidesof the invention, as described above, may further comprise an additionalpolypeptide sequence that enhances solubility, such as an Ig polypeptidesequence, thereby forming, e.g., a soluble fusion protein.

Such polypeptides and dimers of the invention are expected to be ofbeneficial use in a variety of applications, including methods fortreating autoimmune diseases and disorders, and methods for inhibitingorgan, cell, or tissue graft transplantation.

As discussed previously, the mature hCTLA-4 protein sequence typicallybegins with the methionine residue at amino acid position 38 of thefull-length hCTLA-4 protein sequence shown in SEQ ID NO:160, and theamino acid residues of the mature hCTLA-4 protein sequence are typicallynumbered beginning with this methionine residue as the first amino acid(i.e., occupying amino acid position 1).

Some mutant CTLA-4 polypeptides of the invention (including monomericand dimeric fusion proteins and multimeric polypeptides) include atleast one amino acid substitution at an amino acid positioncorresponding to an amino acid position in the mature hCTLA-4 proteinsequence that is outside the classical hCTLA-4/hB7-2 binding interface(see, e.g., Schwartz et al., Nature 410:604-608 (2001)), including, butnot limited to, for example, any of amino acid positions 24, 41, 54, 55,56, 64, 65, 70, and 85. In general, one of ordinary skill in the artwould not have predicted that an amino acid substitution at any of theabove-mentioned positions (24, 41, 54, 55, 56, 64, 65, 70, and/or 85) orany combination of one or more substitutions selected from the group ofpositions 24, 41, 54, 55, 56, 64, 65, 70, and 85 would have an abilityto enhance the binding affinity or avidity of hCTLA-4 for hB7-2, wouldhave an enhanced ability to inhibit the interaction of CD28-positivewith B7-2-positive cells, or would provide a greater ability to suppressor inhibit an immune response than, e.g., hCTLA-4 ECD or hCTLA-4-Ig(e.g., T cell activation or proliferation, cytokine synthesis orproduction, induction of activation markers, synthesis or production ofinflammatory molecules, anti-collagen antibody production, Tcell-dependent antibody response, and the like). Further, one ofordinary skill in the art would not have predicted that a particularamino acid substitution(s) or combination of particular amino acidsubstitutions described herein at any of the above-mentioned positions(24, 41, 54, 55, 56, 64, 65, 70, and/or 85) or any combination of suchpositions would have ability to enhance the binding affinity or avidityof hCTLA-4 for hB7-2, have an enhanced ability to inhibit theinteraction of CD28-positive with B7-2-positive cells, or provide agreater ability to suppress or inhibit an immune response than, e.g.,hCTLA-4 ECD or hCTLA-4-Ig.

Mutant CTLA-4 Fusion Proteins

The invention also provides novel isolated and recombinant fusionproteins which comprise a first polypeptide that is at least one of thepolypeptides of the invention described above and throughout (such as amutant CTLA-4 polypeptide of the invention, such as, e.g., a mutantCTLA-4 ECD polypeptide) linked or fused to a second polypeptide, therebyforming a fusion protein. The second polypeptide typically facilitatessecretion or expression of the first polypeptide. Exemplary mutantCTLA-4 ECD polypeptides include those comprising sequences identified asSEQ ID NOS:1-73. The invention includes fusion proteins comprisingimmunoglobulin (Ig) domains, such as Ig Fc domains, fused or attached tobiological active moieties of the invention, such as mutant CTLA-4polypeptides of the invention. Fusion proteins of the invention arebelieved useful as prophylactic and/or therapeutic agents for theprophylactic and/or therapeutic treatment of a variety of immune systemdiseases and disorders and conditions in immunomodulation and/orimmunosuppression is of benefit, in diagnostic assays, and for thepreparation of medicaments or agents having immunomodulating and/orimmunosuppressive activities or properties as discussed in greaterdetail elsewhere herein.

Fusion proteins of the invention comprising a mutant CTLA-4 polypeptideand an Ig polypeptide (e.g., Ig Fc) are typically termed mutantCTLA-4-Ig fusion proteins. Any of the fusion proteins of the invention,including monomeric and dimeric fusion proteins of the inventiondescribed in greater detail below and in the Examples, may comprise asIg polypeptide, such as, e.g., an Ig Fc polypeptide, as described hereinand elsewhere above and below. The second polypeptide may be linkeddirectly to the first polypeptide. For example, the N-terminus of thesecond polypeptide (e.g., an Ig polypeptide, such as an Ig Fcpolypeptide) may be covalently fused directly to the C-terminus of thefirst polypeptide of the invention (e.g., mutant CTLA-4 ECDpolypeptide). Alternatively, the second polypeptide may be linkedindirectly to the first polypeptide, such as where a linker amino acidsequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acidresidues is included between the first and second polypeptides. Ininstances in which a linker is included, the N-terminus of the aminoacid linker sequence is typically covalently fused to the C-terminus ofthe first polypeptide (e.g., mutant CTLA-4 ECD), and the N-terminus ofthe second polypeptide (e.g., an Ig polypeptide, such as an Ig Fc) istypically covalently fused to the C-terminus of the amino acid linkersequence.

In some instances, the second polypeptide comprises at least a portionof an Ig polypeptide, such as, e.g., one or more domains of an Ig heavychain constant region. The second polypeptide may comprise as a hingeregion, CH2 domain, and CH3 domain of an Ig polypeptide. In someinstances, the second polypeptide comprises an Fc domain of a WT Igpolypeptide (i.e., WT Ig Fc polypeptide), such as, e.g., an Fc domain ofa WT human Ig polypeptide (i.e., WT human Ig Fc polypeptide). Asdiscussed elsewhere, the Ig polypeptide may be from various species,including, e.g., mammal, e.g., human, mouse, non-human primate (e.g.,monkey, gorilla), cat, dog, horse, etc., and can be from various classes(e.g., IgG, IgM, IgE, etc.) and subclasses (e.g., for IgG include IgG1,IgG2, IgG4, etc.), and may comprise an Fc domain or portion of any suchIg polypeptide. The amino acid and nucleic acid sequences of Igpolypeptides of such various species are known in the art.

In one aspect, the invention provides novel isolated or recombinantfusion proteins which each comprise an isolated or recombinant mutantCTLA-4 polypeptide of the invention described above (e.g., mutant CTLA-4ECD) covalently linked or fused, either directly or indirectly (via anamino acid linker sequence), at its C-terminus to the N-terminus of anIg Fc polypeptide, i.e., the Fc domain of an Ig polypeptide. Any of thefusion proteins of the invention, including monomeric and dimeric mutantCTLA-4-Ig fusion proteins of the invention described in greater detailbelow and in the Examples, may comprise as Ig Fc polypeptide asdescribed herein and elsewhere above and below. An Ig Fc polypeptidetypically comprises the hinge region, CH2 domain and CH3 domain of theIg polypeptide. The Ig Fc polypeptide may be derived from variousspecies, including, e.g., human, mouse, primate, etc., and may comprisea wild-type Ig Fc polypeptide (e.g., WT IgG1, IgG2, or IgG4). Exemplaryhuman IgG Fc polypeptides include, e.g., but are not limited to, humanIgG1, human IgG2, human IgG4, etc. The polypeptide sequence of exemplaryhuman IgG1 Fc is set forth in SEQ ID NO:185. The polypeptide sequencesof exemplary human IgG2 Fc polypeptides are set forth in SEQ ID NOS:184and 218, respectively. Alternatively, the Ig Fc polypeptide may comprisea mutant Ig polypeptide. For example, a mutant IgG1 Fc in which one ormore cysteine residues have been substituted with another amino acid(e.g., a serine residue), thereby eliminating one or more disulfidebonds formed between two Ig chains, or in which one or more prolineresidues is substituted with another amino acid (e.g., proline) toreduce effector function (reduced Fc receptor binding), may be includedin a mutant CTLA-4-Ig fusion protein. The polypeptide sequence of anexemplary mutant IgG1 Fc polypeptide is shown in SEQ ID NO:186. Theinvention includes an isolated or recombinant fusion protein, such as amutant CTLA-4-Ig dimer or mutant CTLA-4-Ig monomer, which comprises atleast one recombinant mutant CTLA-4 polypeptide described above linkedat its C-terminus to the N-terminus of a recombinant Ig Fc polypeptidecomprising an amino acid sequence having at least 95%, 96%, 97%, 98%,99%, or 100% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOS:184 (human IgG2 Fc polypeptide), 185 (humanIgG1 Fc polypeptide), 186 (mutant IgG1 Fc polypeptide), and 218 (humanIgG2 Fc polypeptide without C-terminal lysine (K) residue).

In one aspect, the predicted polypeptide sequence of a mutant CTLA-4-Igfusion protein of the invention comprises the following segments: asignal peptide sequence which facilitates secretion of the fusionprotein (e.g., hCTLA-4 signal peptide (SEQ ID NO:182 or SEQ ID NO:216));a mutant CTLA-4 ECD polypeptide, which mutant CTLA-4 ECD polypeptidetypically, but not necessarily, comprises from about 118 to 130 aminoacid residues, and usually about 124 amino acid residues in length; andan Ig Fc polypeptide. Exemplary mutant CTLA-4 ECD polypeptides includethose described above and below. In some instances, no amino acid linkersequence is included between the C-terminus of the mutant CTLA-4 ECDpolypeptide and the N-terminus of the human Ig Fc polypeptide; that is,the C-terminus of a mutant CTLA-4 ECD polypeptide is covalently fuseddirectly to the N-terminus of the Ig Fc polypeptide in the mutantCTLA-4-Ig fusion protein. If desired, however, a mutant CTLA-4-Ig mayinclude a linker (e.g., one or more amino acid residues) between theC-terminus of the mutant CTLA-4 ECD polypeptide and the N-terminus ofthe human Ig Fc polypeptide. The signal peptide of a predicted monomericmutant CTLA-4-Ig fusion protein of the invention is typically cleavedfrom the N-terminus of the mutant CTLA-4 Ig fusion protein duringprocessing and thus the mature or secreted form of a mutant CTLA-4-Igfusion protein of the invention does not usually include a signalpeptide sequence. A fusion protein dimer comprising two such monomericmutant CTLA-4-Ig fusion proteins is typically formed during cellularprocessing by the creation of covalent disulfide bonds between (1)cysteine residues in the mutant CTLA-4 ECD and IgG2 Fc of one suchmonomeric fusion protein and (2) cysteine residues in the mutant CTLA-4ECD and IgG2 Fc of the second (typically, but not necessarily identical)monomeric fusion protein.

The invention includes dimeric fusion proteins (also termed fusionprotein dimer) which each comprise two monomeric fusion proteins of theinvention. The dimer may comprise two identical or different monomericfusion proteins. The dimeric fusion protein is formed by a linkage(s)between the two monomeric fusion proteins. A dimeric fusion proteincomprising two such monomeric fusion proteins is typically formed duringcellular processing by the generation of covalent disulfide bondsbetween cysteine residues in one monomeric fusion protein and cysteineresidues in the second monomeric fusion protein. Thus, in someinstances, a mutant CTLA-4-Ig fusion protein of the invention isexpressed as a dimer comprising two monomeric fusion proteins of theinvention.

In one aspect, the invention provides an isolated or recombinant dimericmutant CTLA-4-Ig fusion protein comprising two monomeric fusionproteins, wherein each monomeric fusion protein comprises a mutantCTLA-4 ECD polypeptide of the invention, as described in detail aboveand further below, fused at its C-terminus to an Ig Fc polypeptide. Thedimer is formed during cellular processing by the generation of covalentdisulfide bonds between cysteine residues in the mutant CTLA-4 ECD andIg Fc of one monomeric fusion protein and cysteine residues in themutant CTLA-4 ECD and Ig Fc of the second monomeric fusion protein. Thetwo monomeric fusion proteins typically, but not necessarily, compriseidentical sequences. The secreted or mature form of a mutant CTLA-4-Igfusion protein does not include a signal peptide, as the signal peptideis typically cleaved from the N-terminus of the protein duringprocessing. The predicted mutant CTLA-4-Ig fusion includes a signalpeptide, the C-terminus of which is typically covalently linked to theN-terminus of the mutant CTLA-4-Ig protein. The N-terminus of eachmonomer of a mature mutant CTLA-4-Ig fusion protein typically comprisesa methionine (M).

As a non-limiting example, the invention provides dimeric fusionproteins comprising two monomeric CTLA-4-Ig fusion proteins, whereineach monomeric mutant CTLA-4-Ig fusion protein comprises a mutant CTLA-4ECD polypeptide linked at its C-terminus to the N-terminus of an Ig Fcpolypeptide, wherein the mutant CTLA-4 ECD polypeptide comprises apolypeptide sequence selected from any of SEQ ID NOS:1-73. In some suchdimeric fusion proteins, the two monomeric fusion proteins arecovalently linked together by a covalent disulfide bond formed duringcellular processing between a cysteine residue at position 120 in eachCTLA-4 mutant ECD polypeptide sequence. Alternatively, or in addition,the two monomeric fusion proteins are covalently linked together by acovalent disulfide bond formed between one or more cysteine residues inthe Ig Fc polypeptide of the first monomeric fusion protein and one ormore cysteine residues in the Ig Fc polypeptide of the second monomericfusion protein. The monomeric fusion proteins may be linked together bymultiple disulfide bonds (e.g., one, two, three, four, or more disulfidebonds) formed during cellular processing between cysteine residuespresent in their respective Ig Fc polypeptides. In some instances, eachmonomeric fusion protein is comprised of the same Ig Fc polypeptide(e.g., human IgG2 Fc, as shown in, e.g., SEQ ID NO:184 or 218), andcovalent disulfide bond(s) may be generated during cellular processingbetween cysteine residues at equivalent positions in each Ig Fcpolypeptide.

An exemplary mutant CTLA-4 ECD polypeptide is the D3-12 mutant CTLA-4ECD polypeptide comprising the polypeptide sequence of SEQ ID NO:11. Anexemplary mutant CTLA-4-Ig fusion protein of the invention is the D3-12mutant CTLA-4 ECD polypeptide covalently linked or fused directly (nolinker) at its C-terminus to the N-terminus of the human IgG2 Fcpolypeptide shown in SEQ ID NO:218, thereby forming the D3-12-IgG2fusion protein shown in SEQ ID NO:205, or covalently linked or fuseddirectly (no linker) at its C-terminus to the N-terminus of the humanIgG2 Fc polypeptide shown in SEQ ID NO:184, thereby forming theD3-12-IgG2 fusion protein shown in SEQ ID NO:74. The sequence of SEQ IDNO:74 differs from that of SEQ ID NO:205 by one residue—i.e., anadditional lysine residue is present at the C-terminus of SEQ ID NO:74.We have found experimentally by liquid chromatography mass spectrometry(LCMS) analysis, or the like, that a mature CTLA-4-Ig fusion proteinmade in CHO cells by transfecting an expression vector comprising anucleotide sequence encoding a mutant CTLA-4 ECD, such as, e.g., theD3-12 ECD polypeptide sequence shown in SEQ ID NO:11, and the hIgG2 Fcpolypeptide shown in SEQ ID NO:184 does not typically include thepredicted C-terminal lysine (K) residue, as would be expected based onthe hIgG2 Fc sequence shown in SEQ ID NO:184.

For example, the nucleotide sequence of SEQ ID NO:153 encodes thehCTLA-4 signal peptide and D3-12-IgG2 fusion protein and includes thestop codon TAA at its C-terminus. The codon AAA, which codes for alysine residue, immediately precedes the stop codon TAA in the sequenceof SEQ ID NO:153. The predicted polypeptide sequence of a matureD3-12-IgG2 fusion protein produced by transfecting an expression vectorcomprising the nucleotide sequence of SEQ ID NO:153 into CHO cells isshown in SEQ ID NO:74. The signal peptide is absent in the mature formof the D3-12-IgG2 fusion protein, as it has been cleaved duringprocessing to form the mature fusion protein. However, we have found,based on LCMS analysis, that in such instance the mature D3-12-IgG2 doesnot typically include the predicted C-terminal lysine residue, as wouldbe expected based on the nucleotide sequence of SEQ ID NO:153. Rather,the resulting mature D3-12-IgG2 polypeptide sequence produced by suchmethod is that shown in SEQ ID NO:205. The C-terminal lysine of the IgG2Fc polypeptide is believed to be cleaved during processing or prior tosecretion.

It is believed that production of D3-12-IgG2 protein using anothermammalian cell line by transfection of such vector comprising thenucleotide sequence of SEQ ID NO:153 into such mammalian cell (e.g., COScells and the like) would produce a similar D3-12-IgG2 fusion proteinlacking the predicted C-terminal lysine residue by virtue of analogousprocessing or secretion machinery.

The dimeric D3-12-IgG2 fusion protein comprises two such D3-12-IgG2monomers linked together by one or more disulfide bonds formed duringcellular processing by the generation of covalent disulfide bondsbetween cysteine residues. D3-12-IgG2 and other fusion proteins of theinvention can be made, e.g., by using methods set forth in Example 3.For example, a nucleic acid sequence encoding a D3-12 polypeptide (e.g.,SEQ ID NO:90) can be cloned into the IgG2 Fc fusion vector, mammaliancells can be transfected with the vector, and the resultant fusionprotein can be expressed (typically in dimeric form), purified, andevaluated as described in Example 3.

Another exemplary mutant CTLA-4 ECD polypeptide is the D3-54 mutantCTLA-4 ECD polypeptide comprising the polypeptide sequence of SEQ IDNO:36, and an exemplary mutant CTLA-4-Ig fusion protein comprises theD3-54 mutant CTLA-4 ECD polypeptide covalently linked or fused directly(no linker) at its C-terminus to the N-terminus of the hIgG2 Fcpolypeptide shown in SEQ ID NO:218 (without the C-terminal lysine),thereby forming the D3-54-IgG2 fusion protein shown in SEQ ID NO:211, orcovalently linked or fused directly (no linker) at its C-terminus to theN-terminus of the hIgG2 Fc polypeptide shown in SEQ ID NO:184 (with theC-terminal lysine), thereby forming the D3-54-IgG2 fusion protein shownin SEQ ID NO:197. As discussed above, experimental analysis indicatesthat the mature D3-54-IgG2 fusion protein made in CHO cells does nottypically include the predicted C-terminal lysine residue. TheC-terminal lysine of hIgG2 Fc is believed to be cleaved duringprocessing or prior to secretion, resulting in the D3-54-IgG2 fusionprotein sequence shown in SEQ ID NO:211. Further, as noted above,D3-29-IgG2 can be made by using methods of Example 3. The dimericD3-54-IgG2 fusion protein comprises two D3-54-IgG2 monomers linkedtogether by one or more disulfide bonds formed during cellularprocessing by the generation of covalent disulfide bonds betweencysteine residues. The nucleic acid sequence shown in SEQ ID NO:201encodes the fusion proteins shown in SEQ ID NOS:197 and 211.

Other fusion proteins of the invention can similarly comprise a mutantCTLA-4 ECD polypeptide linked or fused to hIgG2 (SEQ ID NO:218 or 184).Exemplary mature mutant CTLA-4-IgG2 fusion proteins of the inventioninclude, e.g., the polypeptide sequences of each of SEQ ID NOS:74-79,197-200, 205-214, and 219-222. Each of the polypeptide sequences of SEQID NOS:74-79, 197-200, 220, and 222 includes a C-terminal lysineresidue; this C-terminal lysine residue is typically cleaved duringprocessing or prior to secretion, resulting in the polypeptide sequenceswithout the C-terminal lysine shown in SEQ ID NOS:205-210, 211-214, 219,and 221, respectively.

FIG. 10 is a schematic diagram showing an exemplary configuration orstructure of an exemplary mutant CTLA-4-IgG2 fusion protein of theinvention. Two identical monomeric mutant CTLA-4-IgG2 fusion proteinsare shown schematically, each comprising a mature mutant CTLA-4 ECDpolypeptide covalently linked at its C-terminus to the N-terminus of ahuman IgG2 Fc polypeptide. Each human IgG2 polypeptide includes a humanIgG2 hinge, CH2 domain, and CH3 domain. Exemplary amino acid residuespresent at the junctions between the ECD and Ig Fc polypeptides are alsoshown. The amino acid residues at the junctions between these componentsmay differ depending upon the mutant CTLA-4 ECD polypeptide sequenceand/or Ig polypeptide sequence. This dimeric mutant CTLA-4-IgG2 fusionprotein results from the formation of at least one disulfide bondbetween cysteine residues at analogous positions in the two mutantCTLA-4-IgG2 fusion protein monomers. The cysteine (C) residuespotentially involved in forming disulfide bonds between the two monomersare marked with asterisks. The signal peptide of each monomeric fusionprotein is typically cleaved during processing and thus the secreted(mature) fusion protein typically does not include the signal peptidesequence. The polypeptide sequence of the human IgG2 polypeptide, whichcomprises the hinge, CH2 domain, and CH3 domain of human IgG2, is shownin SEQ ID NO:184. In an alternative aspect, the polypeptide sequence ofthe human IgG2 polypeptide, which comprises the hinge, CH2 domain, andCH3 domain of human IgG2, is shown in SEQ ID NO:218; in this instance,the IgG2 polypeptide does not include the C-terminal lysine (K) residue,as compared to the sequence of SEQ ID NO:184.

The properties of mutant CTLA-4-Ig fusion proteins of the invention,described in detail elsewhere, may be compared to the properties of oneor more reference Ig fusion proteins, such as, e.g., hCTLA-4-IgG1,hCTLA-4-IgG2, Orencia® fusion protein, and LEA29Y-Ig. Properties thatmay be compared include, e.g., ability to bind CD80 and/or CD86 (and/orCD80-Ig and/or CD86-Ig), and/or ability to inhibit or suppress an immuneresponse (e.g., T cell activation or proliferation, cytokine production,etc.). The mature hCTLA-4-IgG1 fusion protein typically exists insolution as a hCTLA-4-IgG1 fusion protein dimer comprising two identicalmonomeric hCTLA-4-IgG1 proteins, each monomeric hCTLA-4-IgG1 fusionprotein comprising a hCTLA-4 ECD polypeptide (SEQ ID NO:159) linked toan IgG1 Fc polypeptide. The mature hCTLA-4-IgG2 fusion protein typicallyexists in solution as an hCTLA-4-IgG2 fusion protein dimer comprisingtwo identical monomeric hCTLA-4-IgG2 proteins, each monomerichCTLA-4-IgG2 fusion protein (SEQ ID NO:162) comprising an hCTLA-4 ECDpolypeptide (SEQ ID NO:159) linked to an IgG2 Fc polypeptide. The matureOrencia® fusion protein is a fusion protein dimer comprising twoidentical monomeric Orencia® fusion proteins, each monomeric fusionprotein (SEQ ID NO:164) comprising a hCTLA-4 ECD polypeptide (SEQ IDNO:159) linked to a specific mutant IgG1 polypeptide (SEQ ID NO:186).The mature LEA29Y-Ig fusion protein typically exists in solution as aLEA29Y-Ig fusion protein dimer comprising two identical monomericLEA29Y-Ig fusion proteins, each monomeric LEA29Y-Ig fusion protein (SEQID NO:166) comprising a specific mutant CTLA-4 ECD polypeptide (SEQ IDNO:168) linked to a specific mutant IgG1 polypeptide (SEQ ID NO:186). Itis believed that the two fusion protein monomers of the Orencia® dimerare covalently linked together by a single disulfide bond formed betweenthe cysteine residue at position 120 of each hCTLA-4-mutant IgG1 monomerand that no disulfide bonds are formed between the two mutant IgG1 Fcpolypeptides.

Some mutant CTLA-4 fusion proteins bind CD80 (e.g., hCD80) and/or CD86(e.g., hCD86). Some such mutant CTLA-4-Ig fusion proteins bind a CD80-Igfusion protein and/or a CD86-Ig fusion protein. Exemplary CD80-Ig fusionproteins include the hCD80-mIg fusion protein (SEQ ID NO:225), whichcomprises a human CD80 ECD linked to a murine Ig Fc polypeptide; and thehCD80-hIgG1 fusion protein (SEQ ID NO:171), which comprises the sequenceof hCD80 ECD linked to human IgG1 Fc polypeptide. Exemplary CD86-Igfusion proteins include the hCD86-mIg fusion protein (SEQ ID NO:226),which comprises an hCD86 ECD (SEQ ID NO:180) linked to a murine Ig Fcpolypeptide; and the mature hCD86-hIgG1 fusion protein (SEQ ID NO:178),which comprises the sequence of hCD86 ECD (SEQ ID NO:180) linked to thehuman IgG1 Fc polypeptide (SEQ ID NO:185). Exemplary nucleic acidsequences encoding hCD86-mIg and hCD80-mIg fusion proteins are shown inSEQ ID NOS:227 and 228, respectively.

In one aspect, the invention provides an isolated or recombinant fusionprotein comprising (a) a polypeptide comprising a polypeptide sequencethat has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5%, or 100% sequence identity to at least one polypeptidesequence selected from the group consisting of SEQ ID NOS:1-73, and (b)an Ig Fc polypeptide (e.g., hIgG2 Fc), wherein the fusion protein bindsCD80 and/or CD86, and/or CD80-Ig and/or CD86-Ig fusion protein, and/orexhibits an ability to inhibit or suppress an immune response. The Ig Fcpolypeptide may comprise a polypeptide sequence having at least 95%,96%, 97%, 98%, 99%, or 100% identity to a polypeptide sequence selectedfrom the group of SEQ ID NO:184, 185, 186, and 218. In some instances,the C-terminus of the polypeptide of (a) is covalently linked to theN-terminus of the Ig Fc polypeptide of (b). Some such mutant CTLA-4-Igfusion proteins bind a mammalian CD80 and/or CD86 (e.g., hCD80 and/orhCD86), and/or a CD80-Ig and/or CD86-Ig fusion protein. A CD80-Ig maycomprise, e.g., a human CD80 ECD linked to an Ig Fc (e.g., hCD80-Ig). Inone embodiment, an hCD80-Ig is a human CD80 ECD linked to a human Ig Fc(hCD80-hIg); in another embodiment, an hCD80-Ig is a human CD80 ECDlinked to a murine Ig Fc (hCD80-mIg). In one embodiment, an hCD86-Ig isa human CD86 ECD linked to a human Ig Fc (hCD86-hIg); in anotherembodiment, an hCD86-Ig is a human CD86 ECD linked to a murine Ig Fc(hCD86-mIg). Some such fusion proteins have an ability to inhibit orsuppress one or more of a variety of immune responses, such as, e.g., Tcell activation, T cell proliferation, cytokine synthesis or production(e.g., production of TNF-α, IFN-γ, IL-2), induction of activationmarkers (e.g., CD25, IL-2 receptor) or inflammatory molecules,inflammation, anti-collagen Ab production, and/or T cell-dependent Abresponse(s) in in vitro and/or in vivo assays and/or methods. Suchfusion proteins are expected to be of beneficial use in a variety ofapplications, including methods for treating immune system diseases anddisorders (e.g., autoimmune diseases), and methods for inhibiting organ,cell, or tissue graft transplantation, as discussed below.

In another aspect, the invention provides an isolated or recombinantmutant CTLA-4-Ig fusion protein dimer comprising two monomeric mutantCTLA-4-Ig fusion proteins linked via at least one disulfide bond formedbetween two cysteine residues present in each monomeric mutant CTLA-4-Igfusion protein. Each mutant CTLA-4-Ig fusion protein monomer comprises:(a) a mutant CTLA-4 ECD polypeptide comprising a polypeptide sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,or 100% sequence identity to at least one polypeptide sequence selectedfrom the group consisting of SEQ ID NOS:1-73, and (b) an Ig Fcpolypeptide (e.g., hIgG2 Fc), wherein the fusion protein dimer bindsCD80 and/or CD86, and/or CD80-Ig and/or CD86-Ig, and/or exhibits anability to inhibit or suppress an immune response. In some instances,the C-terminus of the polypeptide of (a) is covalently linked or fusedto the N-terminus of the Ig Fc polypeptide of (b). The Ig Fc polypeptidemay comprise a polypeptide sequence having at least 95%, 96%, 97%, 98%,99%, or 100% identity to a polypeptide sequence selected from the groupconsisting of SEQ ID NO:184-186 and 218. In some instances, the fusionprotein dimer is formed by a covalent disulfide bond between a cysteineresidue at amino acid position 120 of each mutant CTLA-4 ECD polypeptidesequence, or at an amino acid position corresponding to position 120 ineach mutant CTLA-4 ECD polypeptide sequence relative to the hCTLA-4 ECDpolypeptide sequence shown in SEQ ID NO:159. Some such fusion proteindimers have an ability to inhibit or suppress one or more of a varietyof immune responses, such as, e.g., T cell activation, T cellproliferation, cytokine synthesis or production (e.g., production ofTNF-α, IFN-γ, IL-2), induction of activation markers (e.g., CD25, IL-2receptor) or inflammatory molecules, inflammation, anti-collagen Abproduction, and/or T cell-dependent Ab response(s) in in vitro and/or invivo assays and/or methods. Such fusion protein dimers are expected tobe of beneficial use in a variety of applications, including methods fortreating immune system diseases and disorders (e.g., autoimmunediseases), and methods for inhibiting organ, cell, or tissue grafttransplantation, as discussed below.

Some such mutant CTLA-4-Ig fusion protein monomers have bindingaffinities for hCD86 or hCD86 ECD that are at least equal to or greaterthan those of hCTLA-4 ECD and LEA29 for hCD86 or hCD86 ECD,respectively. See, e.g., Table 5 in Example 4. The mutant CTLA-4 ECDpolypeptide present in some such dimeric and monomeric fusion proteinscomprises a polypeptide sequence having an amino acid length about equalto the amino acid length of the hCTLA-4 ECD. For example, some suchmutant CTLA-4 ECD polypeptides comprise a polypeptide sequence that isabout 110 to 138, 112 to 136, 114 to 134, 116 to 132, 118 to 130, 119 to129, 120 to 128, 121 to 127, 122 to 126, or 123 to 125 amino acidresidues in length. Some such mutant CTLA-4 ECD polypeptides comprise asequence of 124 amino acid residues. Exemplary mutant CTLA-4 ECDpolypeptides include, e.g., but are not limited to, those comprising apolypeptide sequence having at least 95%, 96%, 97%, 98%, 99% or 100%sequence identity to at least one polypeptide sequence selected from thegroup consisting of SEQ ID NOS:1-73, wherein such mutant CTLA-4 ECDpolypeptide binds CD80 and/or CD86 (or an ECD of either or both), and/orhas an ability to inhibit an immune response.

Some such mutant CTLA-4-Ig fusion protein dimers have a binding avidityfor hCD86 and/or hCD86-Ig that is at least about equal to or greaterthan the binding avidity of a hCTLA-4-Ig fusion protein dimer (e.g.,hCTLA-4-IgG2 or hCTLA-4-IgG1 dimer), Orencia® dimer, and/or LEA29Y-Igdimer for hCD86 and/or hCD86-Ig, respectively. Some such fusion proteindimers have a binding avidity for hCD86 and/or hCD86-mIg that is 2-10times (2×-10×), 5-10 times (5×-10×), 10-20 times (10×-20×), 20-40 times(20×-40×), or more than 40 times (>40×) greater than the binding avidityof Orencia® dimer for hCD86 and/or hCD86-mIg. See, e.g., exemplarydimeric fusion proteins of the invention in Table 3 below. Alternativelyor additionally, some such fusion protein dimers have a binding avidityfor hCD80 and/or hCD80-Ig that is at least about equal to or greaterthan the binding avidity of a hCTLA-4-Ig dimer (e.g., hCTLA-4-IgG2 orhCTLA-4-IgG1 dimer), Orencia® dimer, and/or LEA29Y-Ig dimer for hCD80and/or hCD80-Ig, respectively. Some such fusion protein dimers have abinding avidity for hCD80 and/or hCD80-mIg that is 0.5-2 times(0.5×-2×), 2-4 times (2×-4×), or more than 2 times (>2×) greater thanthe binding avidity of Orencia® dimer for hCD86 and/or hCD86-mIg. See,e.g., exemplary dimeric fusion proteins of the invention in Table 4below.

Some such mutant CTLA-4-Ig fusion protein dimers dissociate from bindinghCD86 and/or hCD86-Ig at a rate that is less than the rate at which ahCTLA-4-Ig dimer (e.g., hCTLA-4-IgG2 or hCTLA-4-IgG1 dimer), Orencia®dimer, and/or LEA29Y-Ig dimer dissociates from binding hCD86 and/orhCD86-Ig, respectively. Some such fusion proteins associate with or bindto hCD86 and/or hCD86-Ig at a rate that is at least equal to or greaterthan the rate at which an hCTLA-4-Ig dimer (e.g., hCTLA-4-IgG2 orhCTLA-4-IgG1 dimer), Orencia® dimer, and/or LEA29Y-Ig dimer associateswith hCD86 and/or hCD86-Ig, respectively. For some such fusion proteindimers, the equilibrium dissociation constant (K_(D)) for the bindingreaction between CD86 (or CD86-Ig) and the fusion protein dimer of theinvention is less than the equilibrium dissociation constant (K_(D)) forthe binding reaction between CD86 (or CD86-Ig) and an hCTLA-4-Ig dimer(e.g., hCTLA-4-IgG2 or hCTLA-4-IgG1 dimer), Orencia® dimer, and/orLEA29Y-Ig dimer. See, e.g., exemplary fusion protein dimers of theinvention in Table 3. For some such fusion protein dimers, theequilibrium dissociation constant (K_(D)) for the binding reactionbetween CD80 (or CD80-Ig) and the fusion protein dimer of the inventionis about equal to or less than the equilibrium dissociation constant(K_(D)) for the binding reaction between CD80 (or CD80-Ig) and ahCTLA-4-Ig dimer (e.g., hCTLA-4-IgG2 or hCTLA-4-IgG1 dimer), Orencia®dimer, or LEA29Y-Ig dimer. See, e.g., exemplary fusion protein dimers ofthe invention in Table 4.

Some such mutant CTLA-4-Ig fusion protein dimers have an ability toinhibit or suppress an immune response (e.g., inhibit T cell activationor proliferation, inhibit cytokine production, etc.) that is at leastabout equal to or greater than the ability of a hCTLA-4-Ig dimer (e.g.,hCTLA-4-IgG2 or hCTLA-4-IgG1 dimer), Orencia® dimer, and/or LEA29Y-Igdimer to inhibit or suppress said immune response, respectively. Forexample, some such fusion protein dimers are capable of inhibiting Tcell activation or T cell proliferation in in vitro assays. Examples 4-9set forth below, for example, demonstrate the ability of representativefusion protein dimers of the invention comprising a representativemutant CTLA-4 ECD polypeptide sequence to inhibit T cell proliferationin vitro. Some such dimers are capable of inhibiting or suppressing animmune response in a subject in vivo, such as through the administrationof a therapeutically or prophylactically effective amount of at leastone such dimer to a subject needing immunosuppressive therapy. Some suchfusion protein dimers are expected to be useful in a variety ofapplications, including, e.g., but not limited to, in prophylacticand/or therapeutic methods for inhibiting or suppressing an immuneresponse in a subject suffering from an immune system disease ordisorder in which immunosuppression is desirable (e.g., autoimmunediseases) and methods for inhibiting rejection of a tissue, cell, ororgan transplant from a donor by a recipient.

Some such dimers have varied abilities to modulate or suppress signalingthrough CD28, since they have different comparative binding aviditiesfor CD80 and CD86. Such dimers are useful in applications in whichdifferential manipulation of T cell responses is desirable, includingtherapeutic and prophylactic methods for treating immune system diseasesand disorders, such as, e.g., immunodeficiency diseases and disorders(e.g., RA, MS, psoriasis, etc.). Exemplary dimeric fusion proteinscomprising polypeptides of the invention having some of theabove-described differential CD80/CD86 binding avidities andimmunoinhibitory properties are shown in Example 4.

Some such mutant CTLA-4-Ig dimers have an ability to suppress or inhibitan immune response that is at least about equal to or greater than theability of the hCTLA-4 protein or a hCTLA-4-Ig dimer to suppress orinhibit one or more types of immune responses. For example, some suchdimers have an ability to inhibit T cell activation or proliferation inin vitro and/or in vivo assays and/or applications, such as thosedescribed above and below, which is at least about equal to or greaterthan the ability of the hCTLA-4 protein or a hCTLA-4-Ig dimer (e.g.,Orencia®, hCTLA-4-IgG2 dimer, or hCTLA-4-IgG1 dimer) to inhibit T cellactivation or proliferation in such applications. Additionally, somesuch dimers have an ability to inhibit or suppress an immune response(e.g., T cell activation or proliferation, cytokine production, Tcell-dependent antibody response) that is greater than the ability of aLEA29Y-Ig dimer to inhibit or suppress an immune response. Examples 4-9,e.g., compare the ability of representative dimeric fusion proteins ofthe invention comprising a mutant CTLA-4 ECD polypeptide sequence of theinvention to inhibit T cell proliferation in vitro relative to theability of dimeric hCTLA-4-IgG2, Orencia®, and LEAY29-Ig to inhibit Tcell proliferation in vitro. See, e.g., Tables 6-9 below. Some suchdimers have both an ability to bind hCD80 and/or hCD86 (or hCD80-Igand/or hCD86-Ig) and an ability to inhibit or suppress an immuneresponse in an in vitro and/or in vivo assays and/or applications, suchas those described above and in greater detail below (e.g., an in vivomethod in which in a therapeutically or prophylactically effectiveamount of at least one such dimer is administered). Some such dimershave a binding avidity for hCD80 and/or hCD86 (or hCD80-Ig and/orhCD86-Ig) that is at least about equal to or greater than the bindingavidity of hCTLA-4 protein, a dimeric hCTLA-4-Ig (e.g., hCTLA-4-IgG2,Orencia®) or dimeric LEA29Y-Ig for hCD80 and/or hCD86 (or hCD80-Igand/or hCD86-Ig), respectively, and an ability to inhibit an immuneresponse that is at least equal to or greater than the ability ofhCTLA-4 protein, a dimeric hCTLA-4-Ig (e.g., hCTLA-4-IgG2, Orencia®) ordimeric LEA29Y-Ig to inhibit an immune response. Such mutant CTLA-4-Igfusion protein dimers are expected to be useful in a variety ofapplications, including, e.g., prophylactic and/or therapeutic methodsfor treating immune system diseases, disorders, and conditions, asdiscussed in greater detail infra.

In another aspect, the invention provides an isolated or recombinantfusion protein dimer (e.g., mutant CTLA-4-Ig fusion protein dimer)comprising two identical monomeric fusion proteins (e.g., two monomericmutant CTLA-4-Ig fusion proteins), wherein each such monomeric fusionprotein comprises a polypeptide sequence having at least 90%, 90.5%,91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%,97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identity to a polypeptidesequence selected from the group consisting of SEQ ID NOS:74-79,197-200, 205-214, and 219-222, wherein the dimer binds CD80 and/or CD86(and/or a CD80-Ig and/or CD86-Ig, such as hCD80-mIg and/or hCD86-mIg,respectively), and/or has an ability to inhibit an immune response,including those described above and further below. Each of thepolypeptide sequences set forth in SEQ ID NOS:74-79, 197-200, 205-214,and 219-222 is a mature mutant CTLA-4 ECD fused at its C-terminus to theN-terminus of an IgG2 Fc polypeptide, and each such sequence may betermed a mutant CTLA-4-Ig fusion protein. The polypeptide sequence ofeach of SEQ ID NOS:74-79, 197-200, 220, and 222 is identical to SEQ IDNOS:205-214, 219, and 221, except that each of SEQ ID NOS:74-79,197-200, 220, and 222 includes a lysine at the C-terminus.

In another aspect, the invention provides an isolated or recombinantfusion protein monomer comprising a polypeptide sequence having at least90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%,96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identity to apolypeptide sequence comprising amino acid residues 1-351 of any of SEQID NOS:74-79, 197-200, 205-214, and 219-222, wherein the fusion proteinbinds CD80 and/or CD86 (and/or a CD80-Ig and/or CD86-Ig, such ashCD80-mIg and/or hCD86-mIg, respectively), and/or has an ability toinhibit an immune response, including those described above and furtherbelow.

In another aspect, the invention provides an isolated or recombinantfusion protein dimer comprising two identical monomeric fusion proteins,wherein each such monomeric fusion protein comprises a polypeptidesequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identity to a polypeptide sequence comprising amino acidresidues 1-351 of any of SEQ ID NOS:74-79, 197-200, 205-214, and219-222, wherein said fusion protein dimer protein binds CD80 and/orCD86 (and/or a CD80-Ig and/or CD86-Ig, such as hCD80-mIg and/orhCD86-mIg, respectively), and/or has an ability to inhibit an immuneresponse, including those described above and further below.

Also provided is a mutant monomeric CTLA-4-Ig fusion protein comprisinga polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to a polypeptide sequence selectedfrom the group consisting of SEQ ID NOS:74-79, 197-200, 205-214, and219-222, wherein said monomeric fusion protein binds CD80 and/or CD86(and/or a CD80-Ig and/or CD86-Ig, such as hCD80-mIg and/or hCD86-mIg,respectively), and/or has an ability to inhibit an immune response. Somesuch fusion protein monomers and dimers have an ability to inhibit orsuppress one or more immune responses, including, e.g., T cellactivation or proliferation, cytokine synthesis or production (e.g.,production of TNF-α, IFN-γ, IL-2), induction of activation markers(e.g., CD25, IL-2 receptor), inflammation, anti-collagen Ab production,and/or T cell-dependent Ab response(s)) in in vitro and/or in vivoassays and/or methods (e.g., in vivo in a subject suffering from adisease, disorder, or condition in which immunosuppressive therapy wouldbe of benefit and to whom a therapeutically effective amount of suchdimeric fusion protein is administered as discussed in greater detailbelow). Such fusion protein monomers and dimers are expected to beuseful in a variety of applications, including therapeutic and/orprophylactic methods for treating immune system diseases, includingthose discussed below.

In another aspect, the invention provides an isolated or recombinantfusion protein dimer (e.g., mutant CTLA-4-Ig fusion protein dimer)comprising two monomeric fusion proteins (e.g., monomeric mutantCTLA-4-Ig fusion protein), wherein each such monomeric fusion proteincomprises: (1) a polypeptide (e.g., mutant CTLA-4 extracellular domainpolypeptide) which comprises a polypeptide sequence which differs from apolypeptide sequence selected from the group consisting of SEQ IDNOS:1-73 in no more than 6 amino acid residues (e.g., no more than 1, 2,3, 4, 5, or 6 amino acid residues), and wherein the amino acid residuein the polypeptide sequence at position 41, 50, 54, 55, 56, 64, 65, 70,or 85 is identical to the amino acid residue at the correspondingposition of said selected polypeptide sequence (e.g., a polypeptideselected from SEQ ID NOS:1-73), and (2) an Ig Fc polypeptide (e.g., IgG2Fc), wherein the fusion protein dimer binds CD80 and/or CD86 (and/orCD80-Ig and/or CD86-Ig), and/or inhibits an immune response (e.g., Tcell activation or proliferation, cytokine production, induction ofactivation markers or inflammatory molecules, anti-collagen Abproduction, T cell-dependent Ab responses, etc.) in in vitro and/or invivo assays and/or methods as discussed in detail below. The inventionalso includes an isolated or recombinant monomeric fusion protein asdescribed above which binds CD80 and/or CD86 (and/or CD80-Ig and/orCD86-Ig) and/or induces an immune response in vitro or in vivo. In thefusion protein dimer, the two monomeric fusion proteins (e.g., mutantCTLA-4-Ig monomer) are optionally covalently linked together by one ormore disulfide bonds via cysteine residues in each monomer, and the twomonomers are typically identical to one another. In some instances, themutant CTLA-4 ECD polypeptide in such fusion protein dimer or monomerdiffers from the selected polypeptide (e.g., selected from SEQ IDNOS:1-73) in no more than 6 amino acid residues, but the amino acidoccupying position 41, 50, 54, 55, 56, 64, 65, 70, or 85 is identical tothe amino acid residue included at that position in the selectedpolypeptide sequence; that is, an amino acid residue at such positioncannot be deleted or substituted. Some such mutant CTLA-4 ECDpolypeptides in such a fusion protein comprise a polypeptide sequencewhich differs from the selected polypeptide sequence by no more than 6amino acid residues and which includes amino acid residues at positions24, 30, 32, 41, 50, 54, 55, 56, 64, 65, 70, 85, 104 and 106 that areidentical to the amino acid residues at the corresponding positions inthe selected polypeptide sequence. Such mutant CTLA-4 ECD polypeptidecan differ from the selected polypeptide sequence by amino aciddeletion(s), addition(s), and/or amino acid substitution(s). An aminoacid substitution may be a conservative or non-conservativesubstitution. See, e.g., the “Sequence Variation” section. Some suchdimeric fusion proteins have a binding avidity for hCD86 or hCD86-Igthat is at least about equal to or greater than the binding avidity ofhCTLA-4, dimeric hCTLA-4-Ig, dimeric LEA29Y-Ig, or Orencia® protein forhCD86 or hCD86-Ig, respectively. Some such monomeric fusion proteinshave a binding affinity or avidity for hCD86, hCD86-Ig, or hCD86 ECDthat is at least about equal to or greater than the binding affinity oravidity of the monomeric hCTLA-4, monomeric hCTLA-4-Ig, or monomericLEA29Y-Ig for hCD86, hCD86-Ig, or hCD86 ECD, respectively. Alternativelyor additionally, some such dimeric fusion proteins have a bindingavidity for hCD80 or hCD80-Ig that is at least about equal to or greaterthan the avidity of hCTLA-4 or hCTLA-4-Ig for hCD80, respectively.Alternatively or additionally, some such monomeric fusion proteins havea binding affinity or avidity for hCD80, hCD80-Ig, or hCD80 ECD that isat least about equal to or greater than the binding affinity or avidityof monomeric hCTLA-4 or monomeric hCTLA-4-Ig for hCD80, hCD80-Ig, orhCD80 ECD, respectively. In some instances, the mutant CTLA-4 ECDpolypeptide in such fusion protein dimer or monomer comprises apolypeptide sequence having a length about equal to the amino acidlength of the hCTLA-4 ECD, e.g., from about 118-130, 119-129, 120-128,121-127, 122-126, 123-125, or 124 amino acid residues in length. TheN-terminus of the Ig Fc polypeptide (e.g., IgG2 Fc, IgG1 Fc, IgG4 Fc, ora mutant IgG Fc that reduces effector function or Fc receptor binding)may be covalently linked or fused directly or indirectly (via a linkercomprising, e.g., from 1-10 amino acid residues) to the C-terminus ofthe mutant CTLA-4 ECD polypeptide. The Ig Fc polypeptide may comprise apolypeptide sequence having at least 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to a polypeptide sequence selected from the groupconsisting of SEQ ID NOS:184-186 and 218, e.g., any of SEQ ID NO:184,185, 186, and 218.

Some such mutant CTLA-4-Ig fusion protein dimers and monomers arecapable of suppressing one or more of a variety of immune responses,including, e.g., T cell activation, T cell proliferation, cytokinesynthesis or production (e.g., production of TNF-α, IFN-γ, IL-2),induction of activation markers (e.g., CD25, IL-2 receptor) orinflammatory molecules, inflammation, anti-collagen Ab production,and/or T cell-dependent Ab response(s). Some such mutant CTLA-4-Igdimers have a greater ability to inhibit one or more such immuneresponses than hCTLA-4, dimeric hCTLA-4-Ig, or dimeric LEA29Y-Ig.Examples 4-9, e.g., provide data comparing the ability of representativedimeric fusion proteins of the invention comprising a mutant CTLA-4 ECDpolypeptide of the invention to inhibit T cell proliferation in vitrorelative to the ability of a dimeric hCTLA-4-Ig or dimeric LEA29Y-Ig todo so. Some such mutant CTLA-4-Ig monomers have a greater ability toinhibit one or more such immune responses than monomeric hCTLA-4,monomeric hCTLA-4-Ig, or monomeric LEA29Y-Ig. Some such monomers anddimers are capable of inhibiting or suppressing an immune response in asubject in vivo, such as through the administration of a therapeuticallyor prophylactically effective amount of at least one such polypeptide toa subject needing immunosuppressive therapy. Such fusion proteins areexpected to be of beneficial use in a variety of applications, includingmethods for treating a disease, disorder, or condition in whichimmunosuppressive therapy would be of benefit, such as prophylacticand/or therapeutic methods for treating autoimmune diseases anddisorders, and methods for inhibiting organ, cell, or tissue grafttransplantation.

In another aspect, the invention provides an isolated or recombinantprotein dimer (e.g., mutant CTLA-4-Ig fusion protein dimer) comprisingtwo monomeric fusion proteins (e.g., two monomeric mutant CTLA-4-Igfusion proteins), wherein each such monomeric fusion protein comprises:(1) a mutant CTLA-4 extracellular domain (ECD) polypeptide comprising apolypeptide sequence which (a) differs from a polypeptide sequenceselected from the group consisting of SEQ ID NOS:1-73 in no more than 6amino acid residues (e.g., no more than 1, 2, 3, 4, 5, or 6 amino acidresidues), and (b) comprises at least one amino acid substitution at anamino acid position corresponding to position 50, 54, 55, 56, 64, 65,70, or 85 relative to the polypeptide sequence of SEQ ID NO:159; and (2)an Ig Fc polypeptide, wherein the fusion protein dimer binds CD80 and/orCD86 (and/or CD80-Ig and/or CD86-Ig), and/or inhibits an immune response(e.g., T cell activation or proliferation, cytokine production,induction of activation markers, inflammation, anti-collagen antibodyproduction, T cell-dependent antibody response, etc.) in in vitro and/orin vivo assays and/or methods as described in greater detail below. Theinvention also includes an isolated or recombinant monomeric fusionprotein, as described above, which binds CD80 and/or CD86 (and/orCD80-Ig and/or CD86-Ig) and/or induces an immune response in vitro or invivo. In some instances, CD80 is hCD80 and CD86 is hCD86. In the fusionprotein dimer, the two monomeric fusion proteins (e.g., mutant CTLA-4-Igmonomer) are optionally covalently linked together by one or moredisulfide bonds via cysteine residues in each monomer, and the twomonomers are typically identical to one another. The N-terminus of theIg Fc polypeptide (e.g., IgG2 Fc, IgG1 Fc, IgG4 Fc, or a mutant IgG Fcthat reduces effector function or Fc receptor binding) may be covalentlylinked or fused directly or indirectly (via a linker comprising, e.g.,from 1-10 amino acid residues) to the C-terminus of the mutant CTLA-4ECD polypeptide. The Ig Fc polypeptide may comprise a polypeptidesequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a polypeptide sequence selected from the group consisting ofSEQ ID NOS:184-186 and 218.

Some such fusion protein dimers or monomers comprise a mutant CTLA-4 ECDpolypeptide which comprises a polypeptide sequence having a length aboutequal to the amino acid length of the hCTLA-4 ECD, e.g., 118-130,119-129, 120-128, 121-127, 122-126, or 123-125 amino acid residues inlength. Some such mutant CTLA-4 ECD polypeptides in such fusion proteindimer or monomer comprise a polypeptide sequence that is 124 amino acidresidues in length. Some such mutant CTLA-4-ECD polypeptides comprise 2,3, 4, 5, or 6 amino acid substitutions at positions relative to thesequence set forth in SEQ ID NO:159 selected from the group consistingof position 50, 54, 55, 56, 64, 65, 70, and 85. Some such mutant CTLA-4ECD polypeptides further comprise an amino acid substitution at aposition corresponding to position 104 and/or 30 relative to SEQ IDNO:159. Some such mutant CTLA-4 ECD polypeptides comprise at least oneamino acid substitution relative to SEQ ID NO:159 at position 70(optionally S70F), position 64 (optionally S64P), position 50(optionally A50M), position 54 (optionally M54K/V, e.g., M54K), position65 (optionally I65S), position 56 (optionally N56D), position 55(optionally G55E), position 85 (optionally M85A), and/or position 24(optionally A24E/S, e.g., A24E). Any such mutant CTLA-4 ECD polypeptidemay further comprise an amino acid substitution relative to SEQ IDNO:159 at position 104 (optionally L104E/D, e.g., L104E), position 30(optionally T30N/D/A, e.g., T30N, T30D, or T30A), and/or position 32(optionally V32I). Some such mutant CTLA-4 ECD polypeptides comprise atleast one substitution at an amino acid position relative to SEQ IDNO:159 selected from the group consisting of A50M, M54K, G55E, N56D,S64P, I65S, and S70F. Some such mutant CTLA-4 ECD polypeptides comprise2, 3, 4, 5, or 6 substitutions at amino acid positions relative to SEQID NO:159 selected from the group consisting of A50M, M54K, G55E, N56D,S64P, I65S, and S70F.

Some such mutant CTLA-4-Ig dimers exhibit a binding avidity for CD86(e.g., hCD86) or dimeric CD86-Ig (e.g., hCD86-Ig) that is about equal toor greater than the binding avidity of hCTLA-4 protein, dimerichCTLA-4-Ig (e.g., CTLA-4-IgG1 or CTLA-4-IgG2), Orencia® protein, ordimeric LEA29Y-Ig for CD86 or dimeric CD86-Ig, respectively. Some suchdimers have a binding avidity for CD80 (e.g., hCD80) or dimeric CD80-Ig(e.g., hCD80-Ig) that is greater than the binding avidity of hCTLA-4, adimeric hCTLA-4-Ig, Orencia® protein, and/or dimeric LEAY29-Ig for CD80or dimeric CD80-Ig, respectively.

Some such mutant CTLA-4-Ig monomers exhibit a binding affinity oravidity for CD86 (e.g., hCD86) or CD86-Ig (e.g., hCD86-Ig) that is aboutequal to or greater than the binding affinity or avidity of monomerichCTLA-4, monomeric hCTLA-4-Ig, or monomeric LEA29Y-Ig for CD86 orCD86-Ig, respectively. Some such monomers have a binding affinity oravidity for CD80 (e.g., hCD80) or CD80-Ig (e.g., hCD80-Ig) that isgreater than the binding affinity or avidity of monomeric hCTLA-4 ormonomeric hCTLA-4-Ig (e.g., monomeric CTLA-4-IgG1 or CTLA-4-IgG2) forCD80 or dimeric CD80-Ig, respectively.

Some such mutant CTLA-4-Ig dimers and monomers have an ability tosuppress or inhibit one or more immune responses, including thosedescribed above and throughout (e.g., T cell activation orproliferation, cytokine production, induction of activation markers,inflammation, anti-collagen antibody production, T cell-dependentantibody responses), in in vitro and/or in vivo assays and/or methods(e.g., in vivo in a subject suffering from a disease, disorder, orcondition in which immunosuppressive therapy would be of benefit and towhom a therapeutically effective amount of at least one such mutantCTLA-4-Ig dimer is administered). Some such mutant CTLA-4-Ig dimersinhibit one or more such immune responses to a greater degree thanhCTLA-4, a dimeric hCTLA-4-Ig (e.g., dimeric CTLA-4-IgG1 orCTLA-4-IgG2), Orencia® protein, and/or dimeric LEAY29-Ig. Some suchmutant CTLA-4-Ig monomers inhibit one or more such immune responses to agreater degree than monomeric hCTLA-4, monomeric hCTLA-4-Ig, and/ormonomeric LEAY29-Ig. Such mutant CTLA-4-Ig dimers and monomers areexpected to be of beneficial use in a variety of applications, includingmethods for treating autoimmune diseases and disorders, and methods forinhibiting organ, cell, or tissue graft transplantation.

In another aspect, the invention provides an isolated or recombinantfusion protein dimer (e.g., mutant CTLA-4-Ig fusion protein dimer)comprising two monomeric fusion proteins (e.g., monomeric mutantCTLA-4-Ig fusion protein), wherein each such monomeric fusion proteincomprises: (1) a polypeptide (e.g., mutant CTLA-4 extracellular domain)comprising a polypeptide sequence which (i) has at least 95%, 96%, 97%,98%, 99%, or 100% sequence identity to any polypeptide sequence selectedfrom the group consisting of SEQ ID NOS:1-73 and (ii) includes aphenylalanine residue at an amino acid position corresponding toposition 70 of said polypeptide sequence selected from the groupconsisting of SEQ ID NO:1-73; and (2) an Ig Fc polypeptide (e.g., IgG2Fc, IgG1 Fc, IgG4 Fc, or a mutant IgG Fc that reduces effector functionor Fc receptor binding), wherein the fusion protein dimer binds CD80(e.g., hCD80) and/or CD86 (e.g., hCD86) (and/or CD80-Ig, e.g., hDC80-Ig,and/or CD86-Ig, e.g., hCD86-Ig), and/or has an ability to inhibit animmune response in vitro or in vivo. The invention also includes anisolated or recombinant monomeric fusion protein as described abovewhich binds CD80 (e.g., hCD80) and/or CD86 (e.g., hCD86) (and/orCD80-Ig, e.g., hDC80-Ig, and/or CD86-Ig, e.g., hCD86-Ig) and/or inducesan immune response in vitro or in vivo. In some instances, the Ig Fcpolypeptide comprises a sequence having at least 95%, 96%, 97%, 98%,99%, or 100% sequence identity to a polypeptide sequence selected fromthe group consisting of SEQ ID NOS:184-186 and 218. The N-terminus ofthe Ig Fc polypeptide may be covalently linked or fused directly orindirectly (via a linker comprising, e.g., from 1-10 amino acids) to theC-terminus of the mutant CTLA-4 ECD polypeptide.

In some such mutant CTL-4-Ig dimers or monomers, the mutant CTLA-4 ECDpolypeptide comprises one or more of the following relative to saidselected polypeptide sequence: a glutamic acid residue at an amino acidposition corresponding to position 24; an asparagine residue at an aminoacid position corresponding to position 30; an isoleucine residue at anamino acid position corresponding to position 32; a methionine residueat an amino acid position corresponding to position 50; a lysine residueat an amino acid position corresponding to position 54; a glutamic acidresidue at an amino acid position corresponding to position 55; anaspartic acid residue at an amino acid position corresponding toposition 56; a proline residue at an amino acid position correspondingto position 64; a serine residue at an amino acid position correspondingto position 65; and a glutamic acid residue at an amino acid positioncorresponding to position 104. For example, some such mutant CTLA-4 ECDpolypeptides in such mutant CTLA-4-Ig dimers or monomers comprise apolypeptide sequence comprising (i) at least 95%, 96%, 97%, 98%, 99%, or100% sequence identity to the polypeptide sequence of SEQ ID NO:24 and(ii) a phenylalanine residue at an amino acid position corresponding toposition 70 of the polypeptide sequence of SEQ ID NO:24, wherein thefusion protein dimer binds hCD80 and/or hCD86 (and/or hCD80-Ig and/orhCD86-Ig), and/or inhibits an immune response in in vitro and/or in vivoassays and/or methods. Some such mutant CTLA-4 ECD polypeptides in suchmutant CTLA-4-Ig dimers or monomers comprises one or more of thefollowing relative to SEQ ID NO:24: a glutamic acid residue at position24; an asparagine residue at position 30; an isoleucine residue atposition 32; a methionine residue at position 50; a lysine residue atposition 54; a glutamic acid residue at position 55; an aspartic acidresidue at position 56; a proline residue at position 64; a serineresidue at position 65; and a glutamic acid residue at position 104.

Some such mutant CTLA-4-Ig dimers exhibit a binding avidity for CD86(e.g., hCD86) or dimeric CD86-Ig (e.g., hCD86-Ig) that is about equal toor greater than the binding avidity of hCTLA-4, dimeric hCTLA-4-Ig,Orencia® protein, or dimeric LEA29Y-Ig for CD86 or dimeric CD86-Ig,respectively. Some such dimers have a binding avidity for CD80 (e.g.,hCD80) or dimeric CD80-Ig (e.g., hCD80-Ig) that is greater than thebinding avidity of hCTLA-4, a dimeric hCTLA-4-Ig, or Orencia® proteinfor CD80 or dimeric CD80-Ig, respectively.

Some such mutant CTLA-4-Ig monomers exhibit a binding affinity oravidity for CD86 (e.g., hCD86) or CD86-Ig (e.g., hCD86-Ig) that is aboutequal to or greater than the binding affinity or avidity of monomerichCTLA-4, monomeric hCTLA-4-Ig, and/or monomeric LEA29Y-Ig for CD86 orCD86-Ig, respectively. Some such monomers have a binding affinity oravidity for CD80 (e.g., hCD80) or CD80-Ig (e.g., hCD80-Ig) that isgreater than the binding affinity or avidity of monomeric hCTLA-4 ormonomeric hCTLA-4-Ig for CD80 or dimeric CD80-Ig, respectively.

Some such mutant CTLA-4-Ig dimers and monomers have an ability tosuppress or inhibit one or more immune responses (e.g., T cellactivation or proliferation, cytokine production, induction ofactivation markers, inflammation, anti-collagen Ab production, Tcell-dependent Ab responses), in in vitro and/or in vivo assays and/ormethods (e.g., in vivo in a subject suffering from an immune systemdisease, disorder, or condition in which immunosuppressive therapy wouldbe of benefit and to whom a therapeutically effective amount of at leastone such mutant CTLA-4-Ig dimer is administered). Some such mutantCTLA-4-Ig dimers have an ability to suppress or inhibit one or more suchimmune responses to a greater degree than hCTLA-4, a dimeric hCTLA-4-Ig(e.g., dimeric CTLA-4-IgG1 or CTLA-4-IgG2), Orencia® protein, and/ordimeric LEAY29-Ig. Some such mutant CTLA-4-Ig monomers have an abilityto suppress or inhibit one or more such immune responses to a greaterdegree than monomeric hCTLA-4, monomeric hCTLA-4-Ig, and/or monomericLEAY29-Ig. Such mutant CTLA-4-Ig dimers and monomers are expected to beof beneficial use in a variety of therapeutic and/or prophylacticmethods for treating diseases or disorders in which immunosuppressivetreatment would be of benefit, including, e.g., methods for treatingautoimmune diseases and methods for inhibiting rejection of organ, cell,or tissue graft transplant.

In yet another aspect, the invention provides an isolated or recombinantfusion protein dimer (e.g., mutant CTLA-4-Ig fusion protein dimer)comprising two monomeric fusion proteins (e.g., monomeric mutantCTLA-4-Ig fusion proteins), wherein each such monomeric fusion proteincomprises: (1) a polypeptide (e.g., mutant CTLA-4 extracellular domain)comprising a polypeptide sequence which (a) differs from the polypeptidesequence of the human CTLA-4 extracellular domain polypeptide shown inSEQ ID NO:159 in no more than 6 amino acid residues, and (b) comprisesat least one amino acid substitution, wherein said at least amino acidsubstitution comprises S70F, wherein amino acid residue positions arenumbered according to SEQ ID NO:159; and (2) an IgG Fc polypeptide(e.g., IgG2 Fc, IgG1 Fc, IgG4 Fc, or a mutant IgG Fc that reduceseffector function or Fc receptor binding), wherein said dimer bindshCD80 and/or hCD86 (and/or hCD86-Ig and/or hCD86-Ig), and/or inhibits animmune response (e.g., T cell activation or proliferation, cytokineproduction, induction of activation markers, inflammation, anti-collagenantibody production, T cell-dependent antibody response, etc.) in invitro and/or in vivo assays and/or methods as discussed in detail below.The invention also includes an isolated or recombinant monomeric fusionprotein as described above which binds CD80 (e.g., hCD80) and/or CD86(e.g., hCD86) (and/or CD80-Ig, e.g., hDC80-Ig, and/or CD86-Ig, e.g.,hCD86-Ig) and/or induces an immune response in vitro or in vivo. The IgFc polypeptide may comprise a sequence having at least 95%, 96%, 97%,98%, 99%, or 100% identity to a polypeptide sequence selected from thegroup consisting of SEQ ID NOS:184-186 and 218. The N-terminus of the IgFc polypeptide may be covalently linked or fused directly or indirectly(via a linker comprising, e.g., from 1-10 amino acids) to the C-terminusof the mutant CTLA-4 ECD polypeptide. In some such mutant CTLA-4-Igdimers or monomers, the mutant CTLA-4 ECD polypeptide further comprisesat least one amino acid substitution selected from the group consistingof A24E, T30N, V32I, D41G, A50M, M54K, G55E, N56D, S64P, I65S, M85A,L104E, and I106F. In some such mutant CTLA-4-Ig dimers or monomers, themutant CTLA-4 ECD polypeptide further comprises the substitution L104Eand/or two, three, or four additional substitutions selected from thegroup of substitutions: T30N, V32I, A50M, M54K, G55E, N56D, S64P, andI65S.

Some such dimers have a binding avidity for CD86 (e.g., hCD86) ordimeric CD86-Ig (e.g., hCD86-Ig) that is about equal to or greater thanthe binding avidity of hCTLA-4, dimeric hCTLA-4-Ig, and/or Orencia®protein for CD86 or dimeric CD86-Ig, respectively. Some such dimers havea binding avidity for CD80 (e.g., hCD80) or dimeric CD80-Ig (e.g.,hCD80-Ig) that is greater than the binding avidity of hCTLA-4, a dimerichCTLA-4-Ig, and/or Orencia® for CD80 or dimeric CD80-Ig, respectively.Some such monomers exhibit a binding affinity or avidity for CD86 (e.g.,hCD86) or CD86-Ig (e.g., hCD86-Ig) that is about equal to or greaterthan the binding affinity or avidity of monomeric hCTLA-4, monomerichCTLA-4-Ig, or monomeric LEA29Y-Ig for CD86 or CD86-Ig, respectively.Some such monomers have a binding affinity or avidity for CD80 (e.g.,hCD80) or CD80-Ig (e.g., hCD80-Ig) that is greater than the bindingaffinity or avidity of monomeric hCTLA-4 or monomeric hCTLA-4-Ig forCD80 or dimeric CD80-Ig, respectively.

Some such mutant CTLA-4-Ig dimers and monomers have an ability tosuppress or inhibit one or more immune responses (e.g., T cellactivation or proliferation, cytokine production, induction ofactivation markers, inflammation, anti-collagen antibody production, Tcell-dependent antibody responses), in in vitro and/or in vivo assaysand/or methods (e.g., in vivo in a subject suffering from a disease,disorder, or condition in which immunosuppressive therapy would be ofbenefit and to whom a therapeutically effective amount of at least onesuch mutant CTLA-4-Ig dimer is administered). Some such dimers have anability to suppress or inhibit one or more such immune responses to agreater degree than hCTLA-4, a dimeric hCTLA-4-Ig (e.g., dimericCTLA-4-IgG1 or CTLA-4-IgG2), Orencia® protein, and/or dimeric LEAY29-Ig.Some such monomers have an ability to suppress or inhibit one or moresuch immune responses to a greater degree than monomeric hCTLA-4,monomeric hCTLA-4-Ig, and/or monomeric LEAY29-Ig. Such mutant CTLA-4-Igdimers and monomers are expected to be of beneficial use in a variety oftherapeutic and/or prophylactic methods for treating diseases ordisorders in which immunosuppressive treatment would be of benefit,including, e.g., methods for treating autoimmune diseases and disordersand methods for inhibiting organ or tissue graft transplantation.

In another aspect, the invention provides an isolated or recombinantfusion protein dimer (e.g., mutant CTLA-4-Ig fusion protein dimer)comprising two monomeric fusion proteins (e.g., mutant CTLA-4-Ig fusionprotein), wherein each such monomeric fusion protein comprises: (1) apolypeptide (e.g., mutant CTLA-4 extracellular domain) comprising apolypeptide sequence which (a) differs from the polypeptide sequence ofSEQ ID NO:31 in no more than 6 amino acid residues, and (b) comprises atleast one of the following: a methionine residue at a positioncorresponding to position 50 of SEQ ID NO:31, a lysine residue at aposition corresponding to position 54 of SEQ ID NO:31, a glutamic acidresidue at a position corresponding to position 55 of SEQ ID NO:31, aproline residue at a position corresponding to position 64 of SEQ IDNO:31, a serine residue at a position corresponding to position 65 ofSEQ ID NO:31, a phenylalanine residue at a position corresponding toposition 70 of SEQ ID NO:31, wherein amino acid residue positions arenumbered according to SEQ ID NO:31; and (2) an Ig Fc polypeptide,wherein said dimer binds hCD80 and/or hCD86 (and/or hCD86-Ig and/orhCD86-Ig), and/or inhibits an immune response. The invention alsoincludes an isolated or recombinant monomeric fusion protein asdescribed above which binds CD80 (e.g., hCD80) and/or CD86 (e.g., hCD86)(and/or CD80-Ig, e.g., hDC80-Ig, and/or CD86-Ig, e.g., hCD86-Ig) and/orinduces an immune response in vitro or in vivo. The Ig Fc polypeptidemay comprise a IgG2 Fc, IgG1 Fc, IgG4 Fc, or a mutant IgG Fc thatreduces effector function or Fc receptor binding. The Ig Fc polypeptidemay comprise a sequence having at least 95%, 96%, 97%, 98%, 99%, or 100%identity to a polypeptide sequence selected from the group consisting ofSEQ ID NOS:184-186 and 218. The N-terminus of the Ig Fc polypeptide maybe covalently linked or fused directly or indirectly (via a linkercomprising, e.g., from 1-10 amino acids) to the C-terminus of the mutantCTLA-4 ECD polypeptide. In some such dimers or monomers, the mutantCTLA-4 ECD polypeptide comprises a glutamic acid residue at a positioncorresponding to position 104, an asparagine acid residue at a positioncorresponding to position 30, and/or an isoleucine residue at a positioncorresponding to position 32 of SEQ ID NO:31.

Some such dimers have a binding avidity for CD86 (e.g., hCD86) ordimeric CD86-Ig (e.g., hCD86-Ig) that is about equal to or greater thanthe binding avidity of hCTLA-4 protein, dimeric hCTLA-4-Ig, Orencia®protein, and/or dimeric LEAY29-Ig for CD86 or dimeric CD86-Ig,respectively. Some such dimers have a binding avidity for CD80 (e.g.,hCD80) or dimeric CD80-Ig (e.g., hCD80-Ig) that is greater than thebinding avidity of hCTLA-4, dimeric hCTLA-4-Ig, and/or Orencia® proteinfor CD80 or dimeric CD80-Ig, respectively. Some such monomers exhibit abinding affinity or avidity for CD86 (e.g., hCD86) or CD86-Ig (e.g.,hCD86-Ig) that is about equal to or greater than the binding affinity oravidity of monomeric hCTLA-4, monomeric hCTLA-4-Ig, or monomericLEA29Y-Ig for CD86 or CD86-Ig, respectively. Some such monomers have abinding affinity or avidity for CD80 (e.g., hCD80) or CD80-Ig (e.g.,hCD80-Ig) that is greater than the binding affinity or avidity ofmonomeric hCTLA-4 or monomeric hCTLA-4-Ig for CD80 or dimeric CD80-Ig,respectively.

Some such mutant CTLA-4-Ig dimers and monomers have an ability tosuppress or inhibit one or more immune responses (e.g., T cellactivation or proliferation, cytokine production, induction ofactivation markers, inflammation, anti-collagen antibody production, Tcell-dependent antibody responses) in vitro and/or in vivo as discussedin detail below. Some such dimers have an ability to suppress one ormore such immune responses to a greater degree than hCTLA-4, a dimerichCTLA-4-Ig, Orencia® protein, and/or dimeric LEAY29-Ig. Some suchmonomers have an ability to suppress or inhibit one or more such immuneresponses to a greater degree than monomeric hCTLA-4 or a monomerichCTLA-4-Ig. Such mutant CTLA-4-Ig dimers and monomers are expected to beof beneficial use in a variety of therapeutic and/or prophylacticmethods for treating immune system diseases or disorders in whichimmunosuppressive treatment would be of benefit (e.g., autoimmunediseases and disorders and methods for inhibiting organ or tissue grafttransplantation).

Any such dimeric or monomeric mutant CTLA-4-Ig fusion protein dimer ormonomer described above may further include a peptide that facilitatessecretion of the fusion protein from a host cell. The peptide isoptionally a signal peptide. The C-terminus of the signal peptide istypically covalently linked to the N-terminus of a fusion protein. Thesignal peptide may comprise an amino acid sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to theamino acid sequence of SEQ ID NO:182 or SEQ ID NO:216. The signalpeptide may comprise an amino acid sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an aminoacid sequence comprising amino acid residues 1-35, 1-36, or 1-37 of SEQID NO:160. Furthermore, as discussed below, any such monomeric ordimeric mutant CTLA-4-Ig fusion protein described above may comprise oneor more of the amino acid residues that are glycosylated or pegylated.

The invention also provides a mature/secreted mutant CTLA-4-IgG2 fusionprotein that is 352 amino acids in length and comprises a mutant CTLA-4ECD polypeptide comprising 124 amino acid residues and a human IgG2 Fcpolypeptide comprising 228 amino acid residues. Exemplary mutant CTLA-4ECD polypeptides include those polypeptides comprising sequencesidentified by any of SEQ ID NOS:1-73. Exemplary mutant CTLA-4-IgG2fusion proteins include those comprising a polypeptide sequenceidentified by any of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222. Ifdesired, the amino acids of a mature mutant CTLA-4-IgG2 fusion proteincan be numbered beginning with the first amino acid residue of themutant CTLA-4-IgG2 (i.e., the first residue of the mutant CTLA-4 ECDpolypeptide). In some aspects, the first residue of the mutantCTLA-4-IgG2 fusion protein (or mutant CTLA-4 ECD) is methionine and thusthe numbering of amino acids of the mutant CTLA-4-IgG2 fusion protein(or mutant CTLA-4 ECD) would begin with methionine (designated as aminoacid residue 1).

The invention also includes isolated or recombinant multimeric fusionproteins comprising two or more mutant CTLA-4-Ig fusion proteinsdescribed above. In some instances, the multimer is a fusion proteindimer comprising two mutant CTLA-4-Ig fusion proteins, which may beidentical fusion proteins (i.e., homodimer) or different fusion proteins(i.e., heterodimer). In some instances, the multimer is a tetramericfusion protein, which comprises four mutant CTLA-4-ECD polypeptides ofthe invention. The tetramer can comprise four identical mutant CTLA-4ECD polypeptides (i.e., homotetramer) or any combination of four mutantCTLA-4 ECD polypeptides of the invention such that all four mutantCTLA-4 ECD polypeptides are not identical (i.e., heterotetramer). Somesuch multimers bind CD80 and/CD86 (and/or hCD80-Ig and/or hCD86-Ig)and/or suppress or inhibit an immune response.

The invention includes soluble forms of any of the polypeptides, fusionproteins, and multimers described above. Also included are soluble formson the conjugate of the invention described below. Soluble molecules ofthe invention—e.g., soluble polypeptides, dimeric fusion proteins,monomeric fusion proteins, multimers, and conjugates of theinvention—are not linked or joined or bound to a cell. Some such solublemolecules may be in solution or are capable of circulating, e.g., in afluid (e.g., in a subject's body). A signal peptide may typically beused to facilitate secretion of such a molecule, but the signal peptideis cleaved during secretion of the molecule from a host cell. Thus, inmost instances, a soluble molecule, such as a soluble polypeptide,dimeric fusion protein, monomeric fusion protein, or multimer, does notinclude a signal peptide. As discussed above, a mutant CTLA-4extracellular domain polypeptide of the invention can be linked to an Igmolecule, including, e.g., a portion of an Ig polypeptide, such as,e.g., an Ig Fc polypeptide, which results in a soluble fusion protein.Thus, in one aspect, the invention includes soluble mutant CTLA-4-Igfusion proteins which comprise any mutant CTLA-4 ECD polypeptide of theinvention as described herein fused or linked to at least a portion ofan Ig polypeptide, such as, e.g., a wild-type Ig Fc (e.g., human IgG2Fc) or mutant Ig Fc polypeptide. Such soluble mutant CTLA-4-Ig fusionproteins may be monomeric or dimeric fusion proteins and include thosemutant CTLA-4-Ig fusion protein monomers and dimers described in detailabove and elsewhere, including in the Examples below. As described indetail above and elsewhere herein, some such soluble monomeric anddimeric fusion proteins may have an ability to bind CD80 and/or CD86and/or an ability to suppress or inhibit an immune response (e.g., Tcell activation or proliferation) in in vitro and/or in vivoapplications.

Such soluble molecules of the invention are expected to be of particularbenefit in a variety of applications, including, e.g., therapeutic andprophylactic methods for treating immune system diseases and disorders(e.g., autoimmune diseases) and prophylactic and therapeutic methods forinhibiting cell, organ or tissue graft transplantation. Solublemolecules of the invention—e.g., soluble recombinant mutant CTLA-4 ECDpolypeptides, monomeric and dimeric mutant CTLA-4-Ig fusion proteins,mutant CTLA-4 ECD conjugates, mutant CTLA-4-Ig conjugates, multimerscomprising mutant CTLA-4 ECD polypeptides or mutant CTLA-4-Ig, multimerscomprising mutant CTLA-4 conjugates or mutant CTLA-4-Ig conjugates ofthe invention—which bind CD80 and/or CD86, when administered to asubject in a therapeutically or prophylactically effective amount,inhibit the interaction between endogenous CD80 and/or CD86 andendogenous CD28, thereby suppressing in the subject an immune systemresponse or immune system attack on the subject's healthy body tissues,organs, and/or cells. In instances where a subject is the recipient ofhealthy body tissues, organs, and/or cells from a donor (e.g., such aswhere the subject recipient has received a donor tissue graft or cell ororgan transplant), such soluble molecules inhibit the interactionbetween endogenous CD80 and/or CD86 and endogenous CD28, therebyinhibiting a harmful response or attack by the subject's immune systemon the healthy body tissues, organs, or cells donated to the subject bythe donor. By suppressing an immune system response or attack on healthybody tissues, the side effects (e.g., pain, joint inflammation, etc.)associated with such immune system response or attack on healthytissues, organs, or cells in the subject can be decreased, and thedamage resulting from such a response or attack can be retarded orprevented.

Methods for measuring binding affinities and avidities of polypeptidesof the invention described above, including, e.g., mutant CTLA-4 ECDpolypeptides, dimeric and monomeric mutant CTLA-4-Ig fusion proteins,and multimers of the invention would be known to those of ordinary skillin the art and include, e.g., but are not limited to, Biacore™technology (GE Healthcare), isothermal titration microcalorimetry(MicroCal LLC, Northampton, Mass.), ELISA, binding affinity phagedisplay methods, and FACS methods. Biacore methods are described indetail in Example 4 below. FACS or other sorting methods are describedin greater detail above and elsewhere herein. Methods for measuringbinding avidities of polypeptides of the invention to hCD80 and/or hCD86by phage ELISA are described in Example 2 below.

Methods for detecting and measuring T cell responses induced bymolecules of the invention (including, e.g., mutant CTLA-4 ECDpolypeptides, dimeric and monomeric mutant CTLA-4-Ig fusion proteins,and multimers of the invention) are well known to those skilled in theart. T cell activation is commonly characterized by physiological eventsincluding, e.g., T cell-associated cytokine synthesis (e.g., IFN-γproduction) and induction of activation markers (e.g., CD25, IL-2receptor). CD4+ T cells recognize their immunogenic peptides in thecontext of MHC class II molecules, whereas CD8+ T cells recognize theirimmunogenic peptides in the context of MHC class I molecules. Exemplarymethods for assessing and measuring the ability of molecules of theinvention described above to inhibit or suppress T cell activationand/or T cell proliferation or to block signaling through CD86 and/orCD80 are described in Examples 5-8 and elsewhere herein.

Polypeptides, monomeric and dimeric fusion proteins, and multimers ofthe invention, including those discussed above, optionally furthercomprise an additional amino acid, such as a methionine, added to theN-terminus and/or a peptide tag for purification or identification.Polypeptides of the invention, including those discussed above,optionally further comprise a polypeptide purification subsequence, suchas, e.g., a subsequence is selected from an epitope tag, a FLAG tag, apolyhistidine sequence, and a GST fusion.

In addition, as discussed in greater detail below, the inventionincludes isolated, recombinant, or synthetic nucleic acids encoding allpolypeptides, fusion proteins, and multimers of the invention describedabove and in additional detail below.

Sequence Identity

As discussed above, in one aspect, the invention includes an isolated orrecombinant polypeptide which comprises a polypeptide sequence having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, or 100% sequence identity to a polypeptide sequenceselected from the group consisting of SEQ ID NOS:1-73, wherein thepolypeptide binds CD80 or CD86 or an extracellular domain of eitherand/or has an ability to suppress or inhibit an immune response. Inanother aspect, as described in detail below, the invention provides anisolated or recombinant nucleic acid comprising a polynucleotidesequence that encodes a polypeptide comprising a polypeptide sequencehaving at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to atleast one polypeptide sequence selected from the group consisting of SEQID NOS:1-73, wherein the polypeptide has an ability to bind CD80 and/orCD86 and/or an ECD thereof, and/or has an ability to suppress an immuneresponse, or a complementary polynucleotide sequence thereof.

The degree to which a sequence (polypeptide or nucleic acid) is similarto another provides an indication of similar structural and functionalproperties for the two sequences. Accordingly, in the context of thepresent invention, sequences that have a similar sequence to any givenexemplar sequence are a feature of the present invention. Sequences thathave percent sequence identities as defined below are a feature of theinvention. A variety of methods of determining sequence relationshipscan be used, including manual alignment and computer assisted sequencealignment and analysis. A variety of computer programs for performingsequence alignment are available, or can be produced by one of skill.

As noted above, the sequences of the nucleic acids and polypeptidesemployed in the subject invention need not be identical, but can besubstantially identical to the corresponding sequence of a nucleic acidof the invention or polypeptide of the invention, respectively. Forexample, polypeptides of the invention can be subject to variouschanges, such as one or more amino acid insertions, deletions, and/orsubstitutions, either conservative or non-conservative, including where,e.g., such changes might provide for certain advantages in their use,such as, in their therapeutic or prophylactic use or administration ordiagnostic application. The nucleic acids of the invention can also besubject to various changes, such as one or more substitutions of one ormore nucleic acids in one or more codons such that a particular codonencodes the same or a different amino acid, resulting in either a silentvariation (e.g., mutation in a nucleotide sequence results in a silentmutation in the amino acid sequence, e.g., when the encoded amino acidis not altered by the nucleic acid mutation) or non-silent variation,one or more deletions of one or more nucleic acids (or codons) in thesequence, one or more additions or insertions of one or more nucleicacids (or codons) in the sequence, cleavage of or one or moretruncations of one or more nucleic acids (or codons) in the sequence.The nucleic acids can also be modified to include one or more codonsthat provide for optimum expression in an expression system (e.g.,bacterial or mammalian), while, if desired, said one or more codonsstill encode the same amino acid(s). Such nucleic acid changes mightprovide for certain advantages in their therapeutic or prophylactic useor administration, or diagnostic application. The nucleic acids andpolypeptides can be modified in a number of ways so long as theycomprise a sequence substantially identical (as defined below) to asequence in a respective nucleic acid or polypeptide of the invention.

The term “identical” or “identity,” in the context of two or morenucleic acid or polypeptide sequences, refers to two or more sequencesthat are the same or have a specified percentage of amino acid residuesor nucleotides that are the same, when compared and aligned for maximumsimilarity, as determined using the sequence comparison algorithmdescribed below or by visual inspection. The “percent sequence identity”(“% identity”) of a subject sequence to a reference (i.e. query)sequence means that the subject sequence is identical (i.e., on an aminoacid-by-amino acid basis for a polypeptide sequence, or anucleotide-by-nucleotide basis for a polynucleotide sequence) by aspecified percentage to the query sequence over a comparison length.

The percent sequence identity (“% sequence identity” or “% identity”) ofa subject sequence to a query sequence can be calculated as follows.First, the optimal alignment of the two sequences is determined using asequence comparison algorithm with specific alignment parameters. Thisdetermination of the optimal alignment may be performed using acomputer, or may be manually calculated, as described below. Then, thetwo optimally aligned sequences are compared over the comparison length,and the number of positions in the optimal alignment at which identicalresidues occur in both sequences are determined, which provides thenumber of matched positions. The number of matched positions is thendivided by the total number of positions of the comparison length(which, unless otherwise specified, is the length of the querysequence), and then multiplying the result by 100, to yield the percentsequence identity of the subject sequence to the query sequence.

With regard to polypeptide sequences, typically one sequence is regardedas a “query sequence” (for example, a polypeptide sequence of theinvention) to which one or more other sequences, i.e., “subjectsequence(s)” (for example, sequences present in a sequence database) arecompared. The sequence comparison algorithm uses the designatedalignment parameters to determine the optimal alignment between thequery sequence and the subject sequence(s). When comparing a querysequence against a sequence database, such as, e.g., GENBANK® database(Genetic Sequence Data Bank; U.S. Department of Health and HumanServices) or GENESEQ® database (Thomson Derwent; also available asDGENE® database on STN), usually only the query sequence and thealignment parameters are input into the computer; optimal alignmentsbetween the query sequence and each subject sequence are returned for upto a specified number of subject sequences.

1. Determining the Optimal Alignment

Two polypeptide sequences are “optimally aligned” when they are alignedusing defined parameters, i.e., a defined amino acid substitutionmatrix, gap existence penalty (also termed gap open penalty), and gapextension penalty, so as to arrive at the highest similarity scorepossible for that pair of sequences. The BLOSUM62 matrix (Henikoff andHenikoff (1992) Proc. Natl. Acad. Sci. USA 89(22):10915-10919) is oftenused as a default scoring substitution matrix in polypeptide sequencealignment algorithms (such as BLASTP, described below). The gapexistence penalty is imposed for the introduction of a single amino acidgap in one of the aligned sequences, and the gap extension penalty isimposed for each residue position in the gap. Unless otherwise stated,alignment parameters employed herein are: BLOSUM62 scoring matrix, gapexistence penalty=11, and gap extension penalty=1. The alignment scoreis defined by the amino acid positions of each sequence at which thealignment begins and ends (e.g., the alignment window), and optionallyby the insertion of a gap or multiple gaps into one or both sequences,so as to arrive at the highest possible similarity score.

While optimal alignment between two or more sequences can be determinedmanually (as described below), the process is facilitated by the use ofa computer-implemented alignment algorithm such as BLAST® (NationalLibrary of Medicine), e.g., BLASTP for polypeptide sequences and BLASTNfor nucleic acid sequences, described in Altschul et al. (1997) NucleicAcids Res. 25:3389-3402, and made available to the public throughvarious sources, such as the National Center for BiotechnologyInformation (NCBI) website. When using a computerized BLAST interface,if the option exists to use a “low complexity filter”, this optionshould be turned off (i.e., no filter).

The optimal alignment between two polypeptide sequences can also bedetermined by a manual calculation of the BLASTP algorithm (i.e.,without aid of a computer) using the same alignment parameters specifiedabove (matrix=BLOSUM62, gap open penalty=11, and gap extensionpenalty=1). To begin, the two sequences are initially aligned by visualinspection. An initial alignment score is then calculated as follows:for each individual position of the alignment (i.e., for each pair ofaligned residues), a numerical value is assigned according to theBLOSUM62 matrix (FIG. 13). The sum of the values assigned to each pairof residues in the alignment is the alignment score. If the twosequences being aligned are highly similar, often this initial alignmentprovides the highest possible alignment score. The alignment with thehighest possible alignment score is the optimal alignment based on thealignment parameters employed.

Examples of the manual calculation of alignment scores for two sequencesare provided in FIGS. 14A-14D. FIG. 14A shows is the calculation of analignment score for an arbitrary alignment (alignment 14A) of a “query”sequence, identified herein as residues 39-53 of the human CTLA-4 ECDsequence (SEQ ID NO:159), and a “subject” sequence, identified herein asresidues 40-54 of D3 (SEQ ID NO:61). The numerical value assigned by theBLOSUM62 matrix for each aligned pair of amino acids is shown beneatheach position in the alignment.

FIG. 14B shows the alignment score for the optimal alignment of the sametwo sequences. To aid in visualization, each identical pair of aminoacids in the alignment is shown in boldface. The alignment in FIG. 14B(alignment 14B) below results in the highest possible alignment score(the sum of the values shown beneath each aligned position) of these twosequences; any other alignment of these two sequences, with or withoutgaps, would result in a lower alignment score.

In some instances, a higher alignment score might be obtained byintroducing one or more gaps into the alignment. Whenever a gap isintroduced into an alignment, a gap open penalty is assigned, and inaddition a gap extension penalty is assessed for each residue positionwithin that gap. Therefore, using the alignment parameters describedabove (including gap open penalty=11 and gap extension penalty=1), a gapof one residue in the alignment would correspond to a value of−(11+(1×1))=−12 assigned to the gap; a gap of two residues wouldcorrespond to a value of −(11+(2×1))=−13 assigned to the gap, and so on.This calculation is repeated for each new gap introduced into thealignment.

The following is an example, which demonstrates how introduction of agap into an alignment can result in a higher alignment score, despitethe gap penalty. FIG. 14C shows an alignment (alignment 14C) of a“query” sequence, identified herein as residues 39-53 of the humanCTLA-4 ECD sequence (SEQ ID NO:159), and a “subject” sequence,identified herein as residues 41-55 of D3 (SEQ ID NO:61), but in thisinstance with amino acids 49-50 deleted. Alignment 14C, which is thebest possible alignment without introduction of any gaps, results in analignment score of 34.

The alignment in FIG. 14D (alignment 14D) shows the effect of theintroduction of a two-residue gap in the lower sequence on the alignmentscore. Despite the total gap penalty of 13 (the gap open penalty of 11,and 2 times the gap extension penalty of 1), the overall alignment scoreof the two sequences increases to 43. Alignment D below results in thehighest possible alignment score, and is thus the optimal alignment ofthese two sequences; any other alignment of these two sequences (with orwithout gaps) would result in a lower alignment score.

It is to be understood that the examples of sequence alignmentcalculations described above, which use relatively short sequences, areprovided for illustrative purposes only. In practice, the alignmentparameters employed (BLOSUM62 matrix, gap open penalty=11, and gapextension penalty=1) are generally intended for polypeptide sequences 85amino acids in length or longer. The NCBI website provides the followingalignment parameters for sequences of other lengths, which are suitablefor computer-aided as well as manual alignment calculation, using thesame procedure as described above. For sequences of 50-85 amino acids inlength, optimal parameters are the BLOSUM80 matrix (Henikoff andHenikoff, supra), gap open penalty=10, and gap extension penalty=1. Forsequences of 35-50 amino acids in length, optimal parameters are thePAM70 matrix (Dayhoff, M. O., Schwartz, R. M. & Orcutt, B. C. (1978) “Amodel of evolutionary change in proteins” in Atlas of Protein Sequenceand Structure, vol. 5, suppl. 3, M. O. Dayhoff (ed.), pp. 345-352, Natl.Biomed. Res. Found., Washington, D.C.), gap open penalty=10, and gapextension penalty=1. For sequences of less than 35 amino acids inlength, optimal parameters are PAM30 matrix (Dayhoff, M. O., supra), gapopen penalty=9, and gap extension penalty=1.

2. Calculating Percent Identity

Once the sequences are optimally aligned, the percent identity of thesubject sequence relative to the query sequence is calculated bycounting the number of positions in the optimal alignment which containidentical residue pairs, divide that by the number of residues in thecomparison length (also termed the comparison window), which, unlessotherwise specified, is the number of residues in the query sequence,and multiplying the resulting number by 100. Referring back to thealignments above, in each example the sequence designated as the query(upper) sequence is 15 amino acids in length. In alignment B, 12 pairsof aligned amino acid residues (shown in boldface) are identical in theoptimal alignment of the query sequence (upper) with the subjectsequence (lower). Thus, this particular subject sequence has (12/15)×100=80% identity to the entire length of the 15-residue querysequence; in other words, the subject sequence in alignment B has atleast 80% amino acid sequence identity to the query sequence. Inalignment D, 11 pairs of amino acid residues (shown in boldface) in theoptimal alignment are identical; thus this particular subject sequencehas ( 11/15)×100=73.3% identity to the entire length of the 15-residuequery sequence; in other words, the subject sequence in alignment D hasat least 73% amino acid sequence identity to the query sequence.

As applied to polypeptides, the term “substantial identity” (or“substantially identical”) typically means that when two amino acidsequences (i.e. a query sequence and a subject sequence) are optimallyaligned using the BLASTP algorithm (manually or via computer) usingappropriate parameters described above, the subject sequence has atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5% or 100% amino acid sequence identity tothe query sequence. In some instances, the substantial identity existsover a comparison length of at least 100 amino acid residues, such as,e.g., at least 110, 115, 118, 119, 120, 121, 122, 123, 124, 125, 130,135, 140, 145, 150, 200, 250, 300, 345, 346, 347, 348, 349, 350, 351,352, 353, 354, 355, 356, 357, 358, 359, 360, 375, 400, 450, or 500 aminoacid residues.

Similarly, as applied in the context of two nucleic acid sequences, theterm substantial identity (or substantially identical) means that whentwo nucleic acid sequences (i.e. a query and a subject sequence) areoptimally aligned using the BLASTN algorithm (manually or via computer)using appropriate parameters described below, the subject sequence hasat least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5% or 100% nucleic acid sequence identity to the querysequence. Parameters used for nucleic acid sequence alignments are:match reward 1, mismatch penalty −3, gap existence penalty 5, gapextension penalty 2 (substitution matrices are not used in the BLASTNalgorithm). In some instances, substantial identity exists over acomparison length of at least 300 nucleotide residues, e.g., at least330, 345, 354, 357, 360, 363, 366, 369, 362, 365, 375, 390, 405, 420,435, 450, 600, 750, 900, 1035, 1038, 1041, 1044, 1047, 1050, 1053, 1056,1059, 1062, 1065, 1068, 1071, 1074, 1077, 1080, 1200, 1350, or 1500nucleotide residues.

Other sequence alignment programs known in the art can be used. TheALIGN program produces an optimal global (overall) alignment of the twochosen protein or nucleic acid sequences using a modification of thedynamic programming algorithm described by Myers and Miller CABIOS4:11-17 (1988). The ALIGN program typically, although not necessary, isused with weighted end-gaps. If gap opening and gap extension penaltiesare available, they are often set between about −5 to −15 and 0 to −3,respectively, more preferably about −12 and −0.5 to −2, respectively,for amino acid sequence alignments, and −10 to −20 and −3 to −5,respectively, more commonly about −16 and −4, respectively, for nucleicacid sequence alignments. The ALIGN program is further described inPearson et al., Proc. Natl. Acad. Sci. USA 85:2444-48 (1988), andPearson et al., Meth. Enzymol. 18:63-98 (1990).

Alternatively, and particularly for multiple sequence analysis (i.e.,comparison of more than three sequences), the CLUSTALW program(described in, e.g., Thompson et al., Nucl. Acids Res. 22:4673-4680(1994)) can be used. The CLUSTALW program is an algorithm suitable formultiple DNA and amino acid sequence alignments (Thompson et al., Nucl.Acids Res. 22:4673-4680 (1994)). CLUSTALW performs multiple pairwisecomparisons between groups of sequences and assembles them into amultiple alignment based on homology. In one aspect, Gap open and Gapextension penalties are set at 10 and 0.05, respectively. Alternativelyor additionally, the CLUSTALW program is run using “dynamic” (versus“fast”) settings. Typically, nucleotide sequence analysis with CLUSTALWis performed using the BESTFIT matrix, whereas amino acid sequences areevaluated using a variable set of BLOSUM matrixes depending on the levelof identity between the sequences (e.g., as used by the CLUSTALW version1.6 program available through the San Diego Supercomputer Center (SDSC)or version W 1.8 available from European Bioinformatics Institute,Cambridge, UK). Preferably, the CLUSTALW settings are set to the SDSCCLUSTALW default settings (e.g., with respect to special hydrophilic gappenalties in amino acid sequence analysis). The CLUSTALW program isfurther described in, e.g., Higgins et al., CABIOS 8(2):189-91 (1992),Thompson et al., Nucleic Acids Res. 22:4673-80 (1994), and Jeanmougin etal., Trends Biochem. Sci. 2:403-07 (1998).

In an alternative format, the identity or percent identity between aparticular pair of aligned amino acid sequences refers to the percentamino acid sequence identity that is obtained by CLUSTALW analysis(e.g., version W 1.8), counting the number of identical matches in thealignment and dividing such number of identical matches by the greaterof (i) the length of the aligned sequences, and (ii) 96, and using thefollowing default ClustalW parameters to achieve slow/accurate pairwisealignments—Gap Open Penalty:10; Gap Extension Penalty:0.10; Proteinweight matrix:Gonnet series; DNA weight matrix: IUB; Toggle Slow/Fastpairwise alignments=SLOW or FULL Alignment.

Another useful algorithm for determining percent identity or percentsimilarity is the FASTA algorithm, which is described in Pearson et al.,Proc Natl. Acad. Sci. USA 85:2444 (1988) and Pearson, Methods Enzymol.266:227-258 (1996). Typical parameters used in a FASTA alignment of DNAsequences to calculate percent identity are optimized, BL50 Matrix 15:−5, k-tuple=2; joining penalty=40, optimization=28; gap penalty=−12, gaplength penalty=−2; and width=16.

Other suitable algorithms include the BLAST and BLAST 2.0 algorithms,which facilitate analysis of at least two amino acid or nucleotidesequences, by aligning a selected sequence against multiple sequences ina database (e.g., GenSeq), or, when modified by an additional algorithmsuch as BL2SEQ, between two selected sequences. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (NCBI) (worldwide website addressncbi.nlm.nih.gov). The BLAST algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) can be used with a word length (W) of11, an expectation (E) of 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program (e.g., BLASTP2.0.14; Jun. 29, 2000) can be used with a word length of 3 and anexpectation (E) of 10. The BLOSUM62 scoring matrix (see Henikoff &Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) uses alignments(B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. Again, as with other suitable algorithms, the stringency ofcomparison can be increased until the program identifies only sequencesthat are more closely related to those in the sequence listing herein(e.g., a polypeptide comprising a polypeptide sequence having at least85, 90, 91, 92, 93, 49, 95, 96, 97, 98, 99%, or 100% identity to apolypeptide sequence selected from SEQ ID NOS:1-79, 197-200, 205-214,and 219-222; or nucleic acid comprising a nucleotide sequence having atleast 85, 90, 91, 92, 93, 49, 95, 96, 97, 98, 99%, or 100% identity to anucleotide sequence selected from any of SEQ ID NOS:80-158, 201-204,223, and 224, or a complementary nucleotide sequence thereof.

The BLAST algorithm also performs a statistical analysis of thesimilarity or identity between two sequences (see, e.g., Karlin &Altschul, (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measureof similarity or identity provided by the BLAST algorithm is thesmallest sum probability (P(N)), which provides an indication of theprobability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a nucleic acid isconsidered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, such as less than about 0.01 orless than about 0.001.

BLAST program analysis also or alternatively can be modified by lowcomplexity filtering programs such as the DUST or SEG programs, whichare preferably integrated into the BLAST program operations (see, e.g.,Wootton et al., Comput. Chem. 17:149-63 (1993), Altschul et al., Nat.Genet. 6:119-29 (1991), Hancock et al., Comput. Appl. Biosci. 10:67-70(1991), and Wootton et al., Meth. Enzymol. 266:554-71 (1996)). In suchaspects, if a lambda ratio is used, useful settings for the ratio arebetween 0.75 and 0.95, including between 0.8 and 0.9. If gap existencecosts (or gap scores) are used in such aspects, the gap existence costtypically is set between about −5 and −15, more typically about −10, andthe per residue gap cost typically is set between about 0 to −5, such asbetween 0 and −3 (e.g., −0.5). Similar gap parameters can be used withother programs as appropriate. The BLAST programs and principlesunderlying them are further described in, e.g., Altschul et al., J. Mol.Biol. 215:403-10 (1990), Karlin and Altschul, Proc. Natl. Acad. Sci. USA87:2264-68 (199) (as modified by Karlin and Altschul, Proc. Natl. Acad.Sci. USA 90:5873-77 (1993)), and Altschul et al., Nucl. Acids Res.25:3389-3402 (1997).

Another example of a useful algorithm is incorporated in PILEUPsoftware. The PILEUP program creates a multiple sequence alignment froma group of related sequences using progressive, pair-wise alignments toshow relationship and percent sequence identity or percent sequencesimilarity. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle (1987) J. Mol. Evol. 35:351-360, which issimilar to the method described by Higgins & Sharp (1989) CABIOS5:151-153. The program can align up to 300 sequences, each of a maximumlength of 5,000 nucleotides or amino acids. The multiple alignmentprocedure begins with the pairwise alignment of the two most similarsequences, producing a cluster of two aligned sequences. This cluster isthen aligned to the next most related sequence or cluster of alignedsequences. Two clusters of sequences are aligned by a simple extensionof the pairwise alignment of two individual sequences. The finalalignment is achieved by a series of progressive, pairwise alignments.The program is run by designating specific sequences and their aminoacid or nucleotide coordinates for regions of sequence comparison and bydesignating the program parameters. Using PILEUP, a reference sequenceis compared to other test sequences to determine the percent sequenceidentity (or percent sequence similarity) relationship using specifiedparameters. Exemplary parameters for the PILEUP program are: default gapweight (3.00), default gap length weight (0.10), and weighted end gaps.PILEUP is a component of the GCG sequence analysis software package,e.g., version 7.0 (Devereaux et al. (1984) Nucl. Acids Res. 12:387-395).

Other useful algorithms for performing identity analysis include thelocal homology algorithm of Smith and Waterman (1981) Adv. Appl. Math.2:482, the homology alignment algorithm of Needleman and Wunsch (1970)J. Mol. Biol. 48:443, and the search for similarity method of Pearsonand Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444. Computerizedimplementations of these algorithms (e.g., GAP, BESTFIT, FASTA andTFASTA) are provided in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.

Sequence Variation

As discussed above, in one aspect, the invention provides an isolated orrecombinant mutant CTLA-4 extracellular domain polypeptide whichcomprises a polypeptide sequence which (a) differs from a polypeptidesequence selected from the group consisting of SEQ ID NOS:1-73 in nomore than 6 amino acid residues (e.g., no more than 1, 2, 3, 4, 5, or 6amino acid residues), wherein the mutant CTLA-4 ECD polypeptide bindsCD80 and/or CD86 and/or an extracellular domain of either or both,and/or inhibits an immune response. Such amino acid substitution(s)include conservative amino acid substitution(s).

As a non-limiting example, a polypeptide of the invention may have apolypeptide sequence which differs from SEQ ID NO:1 in a total of up to6 amino acids (which may be a combination of amino acid substitutions,deletions, and/or insertions, including those described above). In someinstances, none, some, or all of the substitutions are substitutionsaccording to a substitution group defined below.

Amino acid substitutions in accordance with the invention may include,but are not limited to, one or more conservative amino acidsubstitutions. A conservative amino acid residue substitution typicallyinvolves exchanging a member within one functional class of amino acidresidues for a residue that belongs to the same functional class(identical amino acid residues are considered functionally homologous orconserved in calculating percent functional homology). Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. One example is provided in Table 1, which sets forthsix exemplary groups containing amino acids that may be considered“conservative substitutions” for one another.

TABLE 1 Conservative Amino Acid Residue Substitution Groups 1 Alanine(A) Glycine (G) Serine (S) Threonine 2 Aspartic acid (D) Glutamic acid(E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R) Lysine (K) Histidine(H) 5 Isoleucine (I) Leucine (L) Methionine (M) Valine (V) 6Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

Other substitution groups of amino acids can be envisioned. For example,amino acids can be grouped by similar function or chemical structure orcomposition (e.g., acidic, basic, aliphatic, aromatic,sulfur-containing). For example, an Aliphatic grouping may comprise:Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I). Othergroups containing amino acids that are considered conservativesubstitutions for one another include: Aromatic: Phenylalanine (F),Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M),Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic:Aspartic acid (D), Glutamic acid (E); Non-polar uncharged residues,Cysteine (C), Methionine (M), and Proline (P); Hydrophilic UnchargedResidues: Serine (S), Threonine (T), Asparagine (N), and Glutamine (Q).See also Creighton (1984) Proteins, W.H. Freeman and Company, foradditional groupings of amino acids. Listing of a polypeptide sequenceherein, in conjunction with the above substitution groups, provides anexpress listing of all conservatively substituted polypeptide sequences.

More conservative substitutions exist within the amino acid residueclasses described above, which also or alternatively can be suitable.Conservation groups for substitutions that are more conservativeinclude: valine-leucine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, and asparagine-glutamine. Thus, forexample, in one particular aspect, the invention provides an isolated orrecombinant polypeptide comprising a polypeptide sequence which has atleast 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1(or any of SEQ ID NOS:1-79, 197-200, 205-214, and 219-222) and whichdiffers from the sequence of SEQ ID NO:1 by mostly (e.g., at least 50%,60%, 70%, 75%, 80%, 90%), if not entirely, by such more conservativeamino acid substitutions.

Additional groups of amino acids substitutions that also can be suitablecan be determined using the principles described in, e.g., Creighton(1984) PROTEINS: STRUCTURE AND MOLECULAR PROPERTIES (2d Ed. 1993), W.H.Freeman and Company. In some aspects, at least 33%, 50%, 60%, 70%, ormore (e.g., at least 75%, 80%, 90%, 95%, 96%, 97% or more) of thesubstitutions in an amino acid sequence variant comprise substitutionsof one or more amino acid residues in a polypeptide sequence of theinvention with residues that are within the same functional homologyclass (as determined by any suitable classification system, such asthose described above) as the amino acid residues of the polypeptidesequence that they replace.

Conservatively substituted variations of a polypeptide sequence of thepresent invention include substitutions of a small percentage, typicallyless than 10%, 9%, 8%, 7%, or 6% of the amino acids of the polypeptidesequence, or more typically less than 5%, 4%, 3%, 2%, or 1%, of theamino acids of the polypeptide sequence, with a conservatively selectedamino acid of the same conservative substitution group.

The invention includes polypeptides that comprise amino acid variationsof a polypeptide sequence of the invention described herein. Asdiscussed above, in one aspect, the invention provides isolated orrecombinant polypeptides (e.g., mutant CTLA-4 polypeptides, such as,e.g., mutant CTLA-4 ECD polypeptides) which each comprise a polypeptidesequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identityto at least one polypeptide sequence selected from the group consistingof SEQ ID NOS:1-73, wherein the polypeptide binds CD80 and/or CD86 or apolypeptide fragment of CD80 and/or CD86 (or an ECD of either or both),and/or suppresses an immune response. Such polypeptides may vary by oneor more amino acid deletions, additions, or substitutions, including oneor more conservative or non-conservative substitutions, provided,however, that the polypeptides possess the described functionalproperties. In a particular aspect, the invention provides polypeptidevariants that comprise conservatively modified variations of any suchpolypeptide described herein, such as, e.g., one comprising apolypeptide sequence selected from the group of SEQ ID NOS:1-73.

As also discussed above, in another aspect, the invention providesisolated or recombinant fusion proteins (e.g., mutant CTLA-4-Ig fusionproteins) which each comprise a polypeptide sequence having at least75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to at least onepolypeptide sequence selected from the group consisting of SEQ IDNOS:74-79, 197-200, 205-214, and 219-222, wherein the fusion proteinbinds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig), and/orsuppresses an immune response. Such fusion proteins may vary by one ormore amino acid deletions, additions, or substitutions, including one ormore conservative or non-conservative substitutions, provided, however,that the fusion proteins possess the described functional properties. Ina particular aspect, the invention provides polypeptide variants thatcomprise conservatively modified variations of any such fusion proteindescribed herein, such as, e.g., one comprising a polypeptide sequenceselected from the group of SEQ ID NOS:74-79, 197-200, 205-214, and219-222.

Also provided are polypeptide variants of any isolated or recombinantpolypeptide of the invention described above or elsewhere herein,wherein the amino acid sequence of the polypeptide variant differs fromthe respective polypeptide sequence of the reference polypeptide by oneor more conservative amino acid residue substitutions, althoughnon-conservative substitutions are sometimes permissible or evenpreferred (examples of such non-conservative substitutions are discussedfurther herein). For example, the sequence of the polypeptide variantcan vary from a mutant CTLA-4 polypeptide sequence by one or moresubstitutions of amino acid residues in the mutant CTLA-4 ECDpolypeptide sequence with one or more amino acid residues having similarweight (i.e., a residue that has weight homology to the residue in therespective polypeptide sequence that it replaces). The weight (andcorrespondingly the size) of amino acid residues of a polypeptide cansignificantly impact the structure of the polypeptide. Weight-basedconservation or homology is based on whether a non-identicalcorresponding amino acid is associated with a positive score on one ofthe weight-based matrices described herein (e.g., BLOSUM50 matrix;PAM250 matrix).

Similar to the above-described functional amino acid classes, naturallyoccurring amino acid residues can be divided into weight-basedconservations groups (which are divided between “strong” and “weak”conservation groups). The eight commonly used weight-based strongconservation groups are Ser Thr Ala, Asn Glu Gln Lys, Asn His Gln Lys,Asn Asp Glu Gln, Gln His Arg Lys, Met Ile Leu Val, Met Ile Leu Phe, HisTyr, and Phe Tyr Trp. Weight-based weak conservation groups include CysSer Ala, Ala Thr Val, Ser Ala Gly, Ser Thr Asn Lys, Ser Thr Pro Ala, SerGly Asn Asp, Ser Asn Asp Glu Gln Lys, Asn Asp Glu Gln His Lys, Asn GluGln His Arg Lys, Phe Val Leu Ile Met, and His Phe Tyr. Some versions ofthe CLUSTAL W sequence analysis program provide an analysis ofweight-based strong conservation and weak conservation groups in theoutput of an alignment, thereby offering a convenient technique fordetermining weight-based conservation (e.g., CLUSTAL W provided by theSDSC, which typically is used with the SDSC default settings). In someaspects, at least 33%, 50%, 60%, 70%, 80%, or 90% of the substitutionsin such polypeptide variant comprise substitutions wherein a residuewithin a weight-based conservation replaces an amino acid residue of thepolypeptide sequence that is in the same weight-based conservationgroup. In other words, such a percentage of substitutions are conservedin terms of amino acid residue weight characteristics.

The sequence of a polypeptide variant can differ from a mutant CTLA-4polypeptide of the invention by one or more amino acid substitutionswith one or more amino acid residues having a similar hydropathy profile(i.e., that exhibit similar hydrophilicity) to the substituted(original) residues of the mutant CTLA-4 polypeptide. A hydropathyprofile can be determined using the Kyte & Doolittle index, the scoresfor each naturally occurring amino acid in the index being as follows: I(+4.5), V (+4.2), L (+3.8), F (+2.8), C (+2.5), M (+1.9); A (+1.8), G(−0.4), T (−0.7), S (−0.8), W (−0.9), Y (−1.3), P (−1.6), H (−3.2); E(−3.5), Q (−3.5), D (−3.5), N (−3.5), K (−3.9), and R (−4.5) (see, e.g.,U.S. Pat. No. 4,554,101 and Kyte & Doolittle, J. Molec. Biol. 157:105-32(1982) for further discussion). At least 75%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98%, 99%, or 100% of the amino acid residues in thevariant polypeptide sequence that are not identical to the correspondingresidues in the identical or functionally homologous mutant CTLA-4polypeptide sequence disclosed herein (“most related homolog”), whichhomolog may be selected from any of SEQ ID NOS:1-73, exhibit less than a+/−2 change in hydrophilicity, including less than a +/−1 change inhydrophilicity and less than a +/−0.5 change in hydrophilicity withrespect to the non-identical amino acid residue at the correspondingposition in the most related homolog. The variant polypeptide mayexhibit a total change in hydrophilicity with respect to its mostrelated homolog selected from the group of SEQ ID NOS:1-73, of less thanabout 150, less than about 100, and/or less than about 50 (e.g., lessthan about 30, 20, or 10).

Examples of typical amino acid substitutions that retain similar oridentical hydrophilicity include arginine-lysine substitutions,glutamate-aspartate substitutions, serine-threonine substitutions,glutamine-asparagine substitutions, and valine-leucine-isoleucinesubstitutions. Algorithms and software, such as the GREASE programavailable through the SDSC, provide a convenient way for quicklyassessing the hydropathy profile of an amino acid sequence. Because asubstantial proportion (e.g., at least about 33%), if not most (at least50%) or nearly all (e.g., about 65, 80, 90, 95, 96, 97, 98, 99%) of theamino acid substitutions in the sequence of a polypeptide variant oftenwill have a similar hydropathy score as the amino acid residue that theyreplace in the (reference) polypeptide sequence, the sequence of thepolypeptide variant is expected to exhibit a similar GREASE programoutput as the polypeptide sequence. For example, in a particular aspect,a polypeptide variant of SEQ ID NO:61 may be expected to have a GREASEprogram (or similar program) output that is more like the GREASE outputobtained by inputting the polypeptide sequence of SEQ ID NO:61 than thatobtained by using a WT CTLA-4 polypeptide (e.g., hCTLA-4), which can bedetermined by visual inspection or computer-aided comparison of thegraphical (e.g., graphical overlay/alignment) and/or numerical outputprovided by subjecting the test variant sequence and SEQ ID NO:1 to theprogram.

The conservation of amino acid residues in terms of functional homology,weight homology, and hydropathy characteristics, also apply to otherpolypeptide sequence variants provided by the invention, including, butnot limited to, e.g., polypeptide sequence variants of a polypeptidesequence selected from the group consisting of SEQ ID NOS:1-79197-200,205-214, and 219-222.

In a particular aspect, the invention includes at least one suchpolypeptide variant comprising an amino acid sequence that differs froma recombinant polypeptide sequence selected from the group of SEQ IDNOS:1-79, 197-200, 205-214, and 219-222, wherein the amino acid sequenceof the variant has at least one such amino acid residue substitutionselected according to weight-based conservation or homology or similarhydropathy profile as discussed above. Such polypeptide variantsdescribed above typically have an ability to bind CD80 and/or CD86and/or an ability to suppress at least one type of immune response asdescribed above and in greater detail below in the Examples.

Signal Peptide Sequences

Polypeptides of the invention can also further comprise any suitablenumber and type of additional amino acid sequences, such as one or morepeptide fragments. In one embodiment, such a polypeptide of theinvention further comprises a signal peptide. Generally, the signalpeptide directs the recombinant polypeptide to the endoplasmic reticulumwhen the recombinant polypeptide is expressed in an animal cell. Asignal sequence that directs organelle trafficking and/or secretion ofat least a portion of the polypeptide upon expression in a cell may beincluded. Such sequences are typically present in the immature (i.e.,not fully processed) form of the polypeptide, and are subsequentlyremoved/degraded by cellular proteases to arrive at the mature form ofthe protein. For example, a mutant CTLA-4 polypeptide or fusion proteinof the invention can include any suitable signal sequence orcombinations of signal sequences that direct the polypeptide tointracellular compartments, such as a sequence that directs thepolypeptide to be transported (e.g., translocated) into (e.g., such thatthe protein is processed by and released from) the endoplasmic reticulumor secretory pathway (e.g., the ER, golgi, and other secretory relatedorganelles and cellular compartments), the nucleus, and/or which directsthe polypeptide to be secreted from the cell, translocated in a cellularmembrane, or target a second cell apart from the cell the protein issecreted from. In this respect, the polypeptide can include anintracellular targeting sequence (or “sorting signal”) that directs thepolypeptide to an endosomal and/or lysosomal compartment(s) or othercompartment rich in MHC II to promote CD4+ and/or CD8+ T cellpresentation and response, such as a lysosomal/endosomal-targetingsorting signal derived from lysosomal associated membrane protein 1(e.g., LAMP-1—see, e.g., Wu et al. Proc. Natl. Acad. Sci. USA 92:1161-75(1995) and Ravipraskash et al., Virology 290:74-82 (2001)), a portion orhomolog thereof (see, e.g., U.S. Pat. No. 5,633,234), or other suitablelysosomal, endosomal, and/or ER targeting sequence (see, e.g., U.S. Pat.No. 6,248,565). In some aspects, it may desirable for the intracellulartargeting sequence to be located near or adjacent to a proven/identifiedepitope sequence(s) within the polypeptide, which can be identified bytechniques known in the art, thereby increasing the likelihood of T cellpresentation of polypeptide fragments that comprise such epitope(s).Such polypeptides may be expressed from an isolated, recombinant, orsynthetic DNA or RNA delivered to a host cell by one or more of thenucleotide transfer vectors, including, e.g., one or more of the genetransfer vectors, described further herein.

The polypeptide may comprise a signal sequence that directs thepolypeptide to the endoplasmic reticulum (ER) (e.g., facilitates ERtranslocation of the polypeptide) when the polypeptide is expressed in amammalian cell. The polypeptide can comprise any suitable ER-targetingsequence. Many ER-targeting sequences are known in the art. Examples ofsuch signal sequences are described in U.S. Pat. No. 5,846,540. Commonlyemployed ER/secretion signal sequences include the yeast alpha factorsignal sequence, and mammalian viral signal sequences such as herpesvirus gD signal sequence. Exemplary signal peptides for E. coliproduction include the STII or Ipp signal sequences of E. coli. Furtherexamples of signal sequences are described in, e.g., U.S. Pat. Nos.4,690,898, 5,284,768, 5,580,758, 5,652,139, and 5,932,445. Suitablesignal sequences can be identified using skill known in the art. Forexample, the SignalP program (described in, e.g., Nielsen et al. (1997)Protein Engineering 10:1-6), which is publicly available through theCenter for Biological Sequence Analysis at the worldwide website addressdesignated cbs.dtu.dk/services/SignalP, or similar sequence analysissoftware capable of identifying signal-sequence-like domains can beused. Related techniques for identifying suitable signal peptides areprovided in Nielsen et al., Protein Eng. 10(1): 1-6 (1997). Sequencescan be manually analyzed for features commonly associated with signalsequences, as described in, e.g., European Patent Application (Appn) No.0 621 337, Zheng and Nicchitta (1999) J. Biol. Chem. 274(51): 36623-30,and Ng et al. (1996) J. Cell Biol. 134(2):269-78.

Additional Aspects

Any polypeptide of the invention (including any fusion protein of theinvention) may be present as part of a larger polypeptide sequence, suchas occurs upon the addition of one or more domains or subsequences forstabilization or detection or purification of the polypeptide. Suchdomains or subsequences may be covalently fused to the polypeptide ofthe invention, as one of skill would readily understand and be able toconstruct. A polypeptide purification subsequence may include, e.g., anepitope tag, a FLAG tag, a polyhistidine sequence, a GST fusion, or anyother detection/purification subsequence or “tag” known in the art.These additional domains or subsequences either have little or no effecton the activity of the polypeptide of the invention, or can be removedby post synthesis processing steps such as by treatment with a protease,inclusion of an intein, or the like.

Any polypeptide of the invention (including any fusion protein of theinvention) may also comprise one or more modified amino acid. Themodified amino acid may be, e.g., a glycosylated amino acid, a PEGylatedamino acid, a farnesylated amino acid, an acetylated amino acid, abiotinylated amino acid, an amino acid conjugated to a lipid moiety,and/or an amino acid conjugated to an organic derivatizing agent. Thepresence of modified amino acids may be advantageous in, for example,(a) increasing polypeptide serum half-life and/or functional in vivohalf-life, (b) reducing polypeptide antigenicity or immunogenicity, (c)increasing polypeptide storage stability, (d) increasingbioavailability, (e) decreasing effector function, and/or (f) decreasingor inhibiting undesired self-association (e.g., aggregate formation)between two or more molecules of the invention (such as between two ormore fusion protein dimers of the invention). Amino acid(s) aremodified, for example, co-translationally or post-translationally duringrecombinant production (e.g., N-linked glycosylation at N-X-S/T motifsduring expression in mammalian cells) or modified by synthetic means.

Polypeptides of the invention (including fusion proteins of theinvention) described herein can be further modified in a variety of waysby, e.g., post-translational modification and/or synthetic modificationor variation. For example, polypeptides or fusion proteins of theinvention may be suitably glycosylated, typically via expression in amammalian cell. For example, in one aspect, the invention providesglycosylated polypeptides that are capable of binding CD86 and/or CD80,and/or have an ability to suppress an immune response (e.g., T cellproliferation or activation) as described elsewhere herein, wherein eachsaid glycosylated polypeptide comprises a polypeptide sequence having atleast 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to asequence selected from the group consisting of SEQ ID NOS:1-79, 197-200,205-214, and 219-222.

The polypeptides of the invention can be subject to any number ofadditional forms suitable of post translational and/or syntheticmodification or variation. For example, the invention provides proteinmimetics of the polypeptides of the invention. Peptide mimetics aredescribed in, e.g., U.S. Pat. No. 5,668,110 and the references citedtherein.

In another aspect, a polypeptide or fusion protein of the invention canbe modified by the addition of protecting groups to the side chains ofone or more the amino acids of the polypeptide or fusion protein. Suchprotecting groups can facilitate transport of the polypeptide or fusionprotein through membrane(s), if desired, or through certain tissue(s),for example, by reducing the hydrophilicity and increasing thelipophilicity of the polypeptide or fusion protein. Examples of suitableprotecting groups include ester protecting groups, amine protectinggroups, acyl protecting groups, and carboxylic acid protecting groups,which are known in the art (see, e.g., U.S. Pat. No. 6,121,236).Synthetic fusion proteins of the invention can take any suitable form.For example, the fusion protein can be structurally modified from itsnaturally occurring configuration to form a cyclic peptide or otherstructurally modified peptide.

Polypeptides of the invention also can be linked to one or morenonproteinaceous polymers, typically a hydrophilic synthetic polymer,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylene, using techniques well known in the art, such asdescribed in, e.g., U.S. Pat. Nos. 4,179,337, 4,301,144, 4,496,689,4,640,835, 4,670,417, and 4,791,192, or a similar polymer such aspolyvinylalcohol or polyvinylpyrrolidone (PVP).

The invention includes conjugates comprising at least one polypeptide ofthe invention (e.g., mutant CTLA-4 ECD polypeptide, dimeric or monomericmutant CTLA-4-Ig, multimeric mutant CTLA-4 ECD polypeptide, multimericmutant CTLA-4-Ig) and a non-polypeptide moiety. The term “conjugate” (orinterchangeably “conjugated polypeptide”) is intended to indicate aheterogeneous (in the sense of composite or chimeric) molecule formed bythe covalent attachment of one or more polypeptide(s) to one or morenon-polypeptide moieties. The term “covalent attachment” means that thepolypeptide and the non-polypeptide moiety are either directlycovalently joined to one another, or else are indirectly covalentlyjoined to one another through an intervening moiety or moieties, such asa bridge, spacer, or linkage moiety or moieties using an attachmentgroup present in the polypeptide. Preferably, the conjugate is solubleat relevant concentrations and conditions, i.e., soluble inphysiological fluids, such as blood. Examples of conjugated polypeptidesof the invention include glycosylated and/or PEGylated polypeptides. Theterm “non-conjugated polypeptide” may be used about the polypeptide partof the conjugate. Such a conjugate typically binds CD80 (e.g., hCD80)and/or CD86 (e.g., hCD86) and/or an extracellular domain of either orboth (including hCD80-Ig and/or hCD86-Ig), and/or has an ability toinhibit an immune response. Such an immune response can comprise, but isnot limited to, e.g., T cell activation or proliferation, cytokinesynthesis/production, induction of activation markers, production ofinflammatory molecules, inflammation, anti-collagen Ab production,and/or T cell-dependent Ab response. Exemplary polypeptides includethose having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to asequence selected from the group of SEQ ID NOS: 1-79, 197-200, 205-214,and 219-222.

The term “non-polypeptide moiety” is intended to indicate a moleculethat is capable of conjugating to an attachment group of a polypeptideof the invention. Preferred examples of such molecule include polymermolecules, sugar moieties, lipophilic compounds, or organic derivatizingagents. When used in the context of a conjugate as described herein itwill be understood that the non-polypeptide moiety is linked to thepolypeptide part of the conjugate through an attachment group of thepolypeptide.

The term “polymer molecule” is defined as a molecule formed by covalentlinkage of two or more monomers, wherein none of the monomers is anamino acid residue, except where the polymer is human albumin or anotherabundant plasma protein. The term “polymer” may be used interchangeablywith the term “polymer molecule”.

An N-glycosylation site has the sequence N-X-S/T/C, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine.

An “O-glycosylation site” comprises the OH-group of a serine orthreonine residue.

The term “attachment group” is intended to indicate an amino acidresidue group of the polypeptide capable of coupling to the relevantnon-polypeptide moiety, such as a polymer molecule or a sugar moiety.Non-limiting examples of useful attachment groups and some correspondingnon-polypeptide moieties are provide in Table 2 below.

TABLE 2 Useful attachment groups and examples of correspondingnon-polypeptide moieties Examples of Examples of non- conjugationAttachment polypeptide method/ group Amino acid moieties activated PEGReference —NH₂ N-terminus, Lys Polymer, e.g., PEG mPEG-SPA Nektar Inc.2003 mPEG2-NHS Catalog; see also mPEG2- Nektar butyrALD Therapeutics,2005-06 Catalog —COOH C-terminus, Asp, Polymer, e.g., PEG mPEG-HzNektar, Inc. Glu Sugar moiety In vitro coupling 2003 Catalog; see alsoNektar Therapeutics 2005-06 Catalog —SH Cys Polymer, e.g., PEG mPEG-VSNektar Inc. 2003 Sugar moiety mPEG2-MAL Catalog; Nektar (mPEG-Therapeutics maleimide) 2005-2006 In vitro coupling Catalog; Delgado etal., Critical Reviews in Therapeutic Drug Carrier Systems 9(3, 4):249-304 (1992) —OH Ser, Thr, OH— Sugar moiety In vivo O-linkedglycosylation —CONH₂ Asn as part of an Sugar moiety In vivo N-N-glycosylation glycosylation site Aromatic Phe, Tyr, Trp Sugar moietyIn vitro coupling residue —CONH₂ Gln Sugar moiety In vitro coupling Yanand Wold, Biochemistry, 1984, Jul. 31; 23(16): 3759-65 Aldehyde OxidizedPolymer, e.g., PEGylation Andresz et al., Ketone carbohydrate PEG, 1978,Makromol. PEG-hydrazide Chem. 179: 301; WO 92/16555, WO 00/23114Guanidino Arg Sugar moiety In vitro coupling Lundblad and Noyes,Chemical Reagents for Protein Modification, CRC Press Inc. Boca Raton,FI Imidazole ring His Sugar moiety In vitro coupling As for guanidine

For in vivo N-glycosylation, the term “attachment group” is used in anunconventional way to indicate the amino acid residues constituting anN-glycosylation site (with the sequence N-X-S/T/C, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine). Although the asparagine residue of theN-glycosylation site is the one to which the sugar moiety is attachedduring glycosylation, such attachment cannot be achieved unless theother amino acid residues of the N-glycosylation site is present.Accordingly, when the non-polypeptide moiety is a sugar moiety and theconjugation is to be achieved by N-glycosylation, the term “amino acidresidue comprising an attachment group for the non-polypeptide moiety”as used in connection with alterations of the amino acid sequence of thepolypeptide of the invention is to be understood as one, two or all ofthe amino acid residues constituting an N-glycosylation site is/are tobe altered in such a manner that either a functional N-glycosylationsite is introduced into the amino acid sequence, removed from saidsequence or a functional N-glycosylation site is retained in the aminoacid sequence (e.g., by substituting a serine residue, which alreadyconstitutes part of an N-glycosylation site, with a threonine residueand vice versa).

The term “introduce” (i.e., an “introduced” amino acid residue,“introduction” of an amino acid residue) is primarily intended to meansubstitution of an existing amino acid residue for another amino acidresidue, but may also mean insertion of an additional amino acidresidue.

The term “remove” (i.e., a “removed” amino acid residue, “removal” of anamino acid residue) is primarily intended to mean substitution of theamino acid residue to be removed for another amino acid residue, but mayalso mean deletion (without substitution) of the amino acid residue tobe removed.

The term “amino acid residue comprising an attachment group for thenon-polypeptide moiety” is intended to indicate that the amino acidresidue is one to which the non-polypeptide moiety binds (in the case ofan introduced amino acid residue) or would have bound (in the case of aremoved amino acid residue).

By removing and/or introducing amino acid residues comprising anattachment group for the non-polypeptide moiety it is possible tospecifically adapt the polypeptide of the invention so as to make themolecule more susceptible to conjugation to the non-polypeptide moietyof choice, to optimize the conjugation pattern (e.g., to ensure anoptimal distribution of non-polypeptide moieties on the surface of thepolypeptide and thereby, e.g., effectively shield epitopes and othersurface parts of the polypeptide without significantly impairing thefunction thereof). For instance, by introduction of attachment groups,the polypeptide is altered in the content of the specific amino acidresidues to which the relevant non-polypeptide moiety binds, whereby amore efficient, specific and/or extensive conjugation is achieved. Byremoval of one or more attachment groups it is possible to avoidconjugation to the non-polypeptide moiety in parts of the polypeptide inwhich such conjugation is disadvantageous, e.g., to an amino acidresidue located at or near a functional site of the polypeptide (sinceconjugation at such a site may result in inactivation or reduced CD80-or CD86-binding or reduced immunosuppressive activity of the resultingconjugate). Further, it may be advantageous to remove an attachmentgroup located close to another attachment group.

The amino acid residue comprising an attachment group for anon-polypeptide moiety, whether an existing residue or a removed orintroduced residue, is selected on the basis of the nature of thenon-polypeptide moiety and, in some instances, on the basis of theconjugation method to be used. For instance, when the non-polypeptidemoiety is a polymer molecule, such as a polyethylene glycol (PEG) orpolyalkylene oxide (POA) derived molecule, amino acid residues capableof functioning as an attachment group may be selected from the groupconsisting of cysteine, lysine (and/or the N-terminal amino group of thepolypeptide), aspartic acid, glutamic acid, histidine and arginine. Whenthe non-polypeptide moiety is a sugar moiety, the attachment group is anin vivo or in vitro N- or O-glycosylation site, preferably anN-glycosylation site.

In some instances, in the mutant CTLA-4 polypeptide part of a conjugateof the invention, attachment groups located at or near the receptorbinding sites are removed, such as by substitution of the amino acidresidue comprising such group. In some instances, amino acid residuescomprising an attachment group for a non-polypeptide moiety, such ascysteine or lysine, are often not introduced at or near the receptorbinding site of the mutant CTLA-4 polypeptide.

A mutant CTLA-4 polypeptide of the invention can be modified so as toshield and thereby modify or destroy or otherwise inactivate an epitopepresent in the mutant CTLA-4 polypeptide, by conjugation to anon-polypeptide moiety. Epitopes of mutant CTLA-4 polypeptides may beidentified by use of methods known in the art, also known as epitopemapping, see e.g., Romagnoli et al., J. Biol. Chem. 380(5):553-9 (1999),DeLisser H M, Methods Mol Biol, 1999, 96:11-20, Van de Water et al.,Clin. Immunol. Immunopathol. 85(3):229-35 (1997), Saint-Remy J M,Toxicology 119(1):77-81 (1997).

The exact number of attachment groups available for conjugation andpresent in the mutant CTLA-4 polypeptide is dependent on the effectdesired to be achieved by conjugation. The effect to be obtained is,e.g., dependent on the nature and degree of conjugation (e.g., theidentity of the non-polypeptide moiety, the number of non-polypeptidemoieties desirable or possible to conjugate to the polypeptide, wherethey should be conjugated or where conjugation should be avoided, etc.).For instance, if reduced immunogenicity is desired, the number (andlocation of) attachment groups should be sufficient to shield most orall epitopes. This is normally obtained when a greater proportion of themutant CTLA-4 polypeptide is shielded. Effective shielding of epitopesis normally achieved when the total number of attachment groupsavailable for conjugation is in the range of 1-6 attachment groups,e.g., 1-5, such as in the range of 1-3, such as 1, 2, or 3 attachmentgroups.

Functional in vivo half-life can be dependent on the molecular weight ofthe conjugate, and the number of attachment groups needed for providingincreased half-life thus depends on the molecular weight of thenon-polypeptide moiety in question. Some such conjugates comprise 1-6,e.g., 1-5, such as 1-3, e.g., 1, 2, or 3 non-polypeptide moieties eachhaving a molecular weight of about 100-2000 Daltons (Da), such as about200 Da, about 300 Da, about 400 Da, about 600 Da, about 900 Da, about1000 Da, or about 2-40 kDa, such as about 2 kDa, about 5 kDa, about 12kDa, about 15 kDa, about 20 kDa, about 30 kDa, about 40 kDa, or about 60kDa.

In the conjugate of the invention, some, most, or substantially allconjugatable attachment groups are occupied by the relevantnon-polypeptide moiety.

The conjugate of the invention may exhibit one or more of the followingimproved properties: (a) increased serum half-life and/or functional invivo half-life, (b) reduced antigenicity or immunogenicity, (c)increased storage stability, (d) increased bioavailability, (e)decreased effector function, or (f) decreased or inhibitedself-association (e.g., decreased aggregate formation) between two ormore molecules of the invention. For example, the conjugate may exhibita reduced immunogenicity as compared to hCTLA-4 or as compared to thecorresponding non-conjugated polypeptide, e.g., a reduction of at least10%, such as a reduction of at least of 25%, such as a reduction of atleast of 50%, e.g., a reduction of at least 75% compared to thenon-conjugated polypeptide or compared to a hCTLA-4. The conjugate mayexhibit an increased functional in vivo half-life and/or increased serumhalf-life as compared to a reference molecule, such as hCTLA-4 or ascompared to the corresponding non-conjugated polypeptide. Particularpreferred conjugates are such conjugates where the ratio between thefunctional in vivo half-life (or serum half-life) of said conjugate andthe functional in vivo half-life (or serum half-life) of said referencemolecule is at least 1.25, such as at least 1.50, such as at least 1.75,such as at least 2, such as at least 3, such as at least 4, such as atleast 5, such as at least 6, such as at least 7, such as at least 8. Thehalf-life is conveniently determined in an experimental animal, such asrat or monkey, and may be based on intravenously or subcutaneouslyadministration. In a further aspect, the conjugate may exhibit anincreased bioavailability as compared to a reference molecule such as anhCTLA-4 or a corresponding non-conjugated polypeptide.

The polymer molecule to be coupled to the polypeptide may be anysuitable polymer molecule, such as a natural or synthetic homopolymer orheteropolymer, typically with a molecular weight in the range of300-100,000 Da, such as 300-20,000 Da, more preferably in the range of500-10,000 Da, even more preferably in the range of 500-5000 Da.

Examples of homopolymers include a polyol (i.e. poly-OH), a polyamine(i.e. poly-NH₂) and a polycarboxylic acid (i.e. poly-COOH). Aheteropolymer is a polymer, which comprises one or more differentcoupling groups, such as, e.g., a hydroxyl group and an amine group.Examples of suitable polymer molecules include polymer moleculesselected from the group consisting of polyalkylene oxide (PAO),including polyalkylene glycol (PAG), such as polyethylene glycol (PEG)and polypropylene glycol (PPG), branched PEGs, poly-vinyl alcohol (PVA),poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleic acidanhydride, polystyrene-co-malic acid anhydride, dextran includingcarboxymethyl-dextran, or any other biopolymer suitable for reducingimmunogenicity and/or increasing functional in vivo half-life and/orserum half-life. Another example of a polymer molecule is human albuminor another abundant plasma protein. Generally, polyalkyleneglycol-derived polymers are biocompatible, non-toxic, non-antigenic,non-immunogenic, have various water solubility properties, and areeasily excreted from living organisms.

PEG is the preferred polymer molecule to be used, since it has only fewreactive groups capable of cross-linking compared, e.g., topolysaccharides such as dextran, and the like. In particular,monofunctional PEG, e.g., monomethoxypolyethylene glycol (mPEG), is ofinterest since its coupling chemistry is relatively simple (only onereactive group is available for conjugating with attachment groups onthe polypeptide). Consequently, the risk of cross-linking is eliminated,the resulting polypeptide conjugates are more homogeneous and thereaction of the polymer molecules with the polypeptide is easier tocontrol. When the molecule is PEGylated, it usually comprises 1, 2, 3,4, or 5 polyethylene glycol (PEG) molecules. Each PEG molecule can havea molecular weight of about 5 kDa (kilo Dalton) to 100 kDa, including,e.g., about 10 kDa, about 12 kDa, about 20 kDa, about 40 kDa. SuitablePEG molecules are available from Shearwater Polymers, Inc. and Enzon,Inc. and may be selected from SS-PEG, NPC-PEG, aldehyde-PEG, mPEG-SPA,mPEG-SCM, mPEG-BTC, SC-PEG, tresylated mPEG (U.S. Pat. No. 5,880,255),or oxycarbonyl-oxy-N-dicarboxylmide-PEG (U.S. Pat. No. 5,122,614).

In one aspect, the invention provides an isolated or synthetic conjugatecomprising: (a) a polypeptide of the invention (e.g., mutant CTLA-4 ECDpolypeptide, dimeric or monomeric mutant CTLA-4-Ig, multimeric mutantCTLA-4 ECD polypeptide, multimeric mutant CTLA-4-Ig); and (b) at leastone non-polypeptide moiety, such as, e.g., 1-10, 1-9, 1-8, 1-7, 1-7,1-6, 1-5, 1-4, 1-3, 1, 2, or 3 non-polypeptide moieties attached to thepolypeptide, wherein the conjugate binds CD80 (e.g., hCD80) and/or CD86(e.g., hCD86) and/or an extracellular domain of either or both(including hCD80-Ig and/or hCD86-Ig), and/or has an ability to induce animmune response (e.g., T cell-dependent immune response). Exemplarypolypeptides include those having at least 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identity to a polypeptide sequence selected from thegroup of SEQ ID NOS:1-79, 197-200, 205-214, and 219-222. In someinstances, the conjugate comprises one non-polypeptide moiety. In someinstances, the conjugate comprises two, three, four or morenon-polypeptide moieties. In some instances, the amino acid sequence ofthe polypeptide of the conjugate further comprises one or moresubstitutions which each introduce an attachment group for thenon-polypeptide moiety (e.g., by substitution of an amino acid residueof the polypeptide sequence with a different residue which comprises anattachment group for the non-polypeptide moiety, or by insertion intothe polypeptide sequence of an additional amino acid residue whichcomprises an attachment group for the non-polypeptide moiety).

A conjugate can comprise two or more polypeptides of the invention. Insome instances, a non-polypeptide moiety is covalently attached toeither or both such polypeptides. If the conjugate comprises two or moreidentical polypeptides of the invention, the same type and number ofnon-polypeptide moieties are typically attached to each suchpolypeptide, usually in the same manner to the corresponding attachmentgroup(s) on each polypeptide. As noted above, the non-polypeptide moietycan comprise, e.g., a sugar molecule, which optionally can be attachedto an N-glycosylation site, or a polymer, such as, e.g., a polyethyleneglycol moiety. The polyethylene glycol moiety can be covalently attachedto a cysteine residue or lysine residue of the polypeptide of theinvention. In some instances, the polyethylene glycol moiety iscovalently attached to the N-terminal amino group of the polypeptide. Aconjugate comprising a mutant CTLA-4-Ig of the invention may bedescribed as a mutant CTLA-4-Ig conjugate of the invention. Multimers ofconjugates are also included. Multimeric conjugates include two or moreconjugates, wherein at least one conjugate is a conjugate of theinvention comprising at least one polypeptide of the invention. Theconjugates in a multimeric conjugate can be, but need not be, identicalto one another.

As discussed above, polypeptides of the invention, including fusionproteins of the invention, can commonly be subject to glycosylation.Polypeptides and fusion proteins of the invention can further be subjectto (or modified such that they are subjected to) other forms ofpost-translational modification including, e.g., hydroxylation, lipid orlipid derivative-attachment, methylation, myristylation, pegylation,phosphorylation, and sulfation. Other post-translational modificationsthat a polypeptide or fusion protein of the invention can be renderedsubject to include acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of phosphotidylinositol, cross-linking, cyclization,disulfide bond formation, demethylation, formylation, GPI anchorformation, iodination, oxidation, proteolytic processing, prenylation,racemization, selenoylation, arginylation, and ubiquitination. Othercommon protein modifications are described in, e.g., Creighton, supra,Seifter et al., Meth. Enzymol. 18:626-646 (1990), and Rattan et al.,Ann. NY Acad. Sci. 663:48-62 (1992). Post-translational modificationsfor polypeptides or fusion proteins expressed from nucleic acids in hostcells vary depending what kind of host or host cell type the peptide isexpressed in. For instance, glycosylation often does not occur inbacterial hosts such as E. coli and varies considerably in baculovirussystems as compared to mammalian cell systems. Accordingly, whenglycosylation is desired (which usually is the case for mostpolypeptides of the present invention), a polypeptide or fusion proteinshould be expressed (produced) in a glycosylating host, generally aeukaryotic cell (e.g., a mammalian cell or an insect cell).Modifications to the polypeptide or fusion protein in terms ofpost-translational modification can be verified by any suitabletechnique, including, e.g., x-ray diffraction, NMR imaging, massspectrometry, and/or chromatography (e.g., reverse phase chromatography,affinity chromatography, or GLC).

The polypeptide or fusion protein also or alternatively can comprise anysuitable number of non-naturally occurring amino acids (e.g., β aminoacids) and/or alternative amino acids (e.g., selenocysteine), or aminoacid analogs, such as those listed in the MANUAL OF PATENT EXAMININGPROCEDURE §2422 (7th Revision—2000), which can be incorporated byprotein synthesis, such as through solid phase protein synthesis (asdescribed in, e.g., Merrifield, Adv. Enzymol. 32:221-296 (1969) andother references cited herein). A polypeptide or fusion protein of theinvention can further be modified by the inclusion of at least onemodified amino acid. The inclusion of one or more modified amino acidsmay be advantageous in, for example, (a) increasing polypeptide orfusion protein serum half-life, (b) reducing polypeptide or fusionprotein antigenicity, or (c) increasing polypeptide or fusion proteinstorage stability. Amino acid(s) are modified, for example,co-translationally or post-translationally during recombinant production(e.g., N-linked glycosylation site at N-X-S/T motifs during expressionin mammalian cells) or modified by synthetic means. Non-limitingexamples of a modified amino acid include a glycosylated amino acid, asulfated amino acid, a prenylated (e.g., farnesylated,geranylgeranylated) amino acid, an acetylated amino acid, an acylatedamino acid, a PEGylated amino acid, a biotinylated amino acid, acarboxylated amino acid, a phosphorylated amino acid, and the like.References adequate to guide one of skill in the modification of aminoacids are replete throughout the literature. Example protocols are foundin Walker (1998) Protein Protocols on CD-ROM Humana Press, Towata, N.J.The modified amino acid may be selected from a glycosylated amino acid,a PEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, an amino acid conjugated to a lipidmoiety, and an amino acid conjugated to an organic derivatizing agent.

The invention further provides polypeptides (including fusion proteins)having the above-described characteristics that further compriseadditional amino acid sequences that impact the biological function(e.g., immunogenicity, targeting, and/or half-life) of the polypeptide(or fusion protein).

A polypeptide or fusion protein of the invention may further include atargeting sequence other than, or in addition to, a signal sequence. Forexample, the polypeptide or fusion protein can comprise a sequence thattargets a receptor on a particular cell type (e.g., a monocyte,dendritic cell, or associated cell) to provide targeted delivery of thepolypeptide to such cells and/or related tissues. Signal sequences aredescribed above, and include membrane localization/anchor sequences(e.g., stop transfer sequences, GPI anchor sequences), and the like.

A particularly useful fusion partner for a polypeptide of the invention(including a fusion protein of the invention) is a peptide sequence thatfacilitates purification of the polypeptide, e.g., a polypeptidepurification subsequence. A polynucleotide of the invention may comprisea coding sequence fused in-frame to a marker amino acid sequence that,e.g., facilitates purification of the encoded polypeptide. Suchpurification facilitating peptide domains or polypeptide purificationsubsequences include, but are not limited to, metal chelating peptides,such as histidine-tryptophan modules that allow purification onimmobilized metals, such as a hexa-histidine peptide or other apolyhistidine sequence, a sequence encoding such a tag is incorporatedin the pQE vector available from QIAGEN, Inc. (Chatsworth, Calif.), asequence which binds glutathione (e.g., glutathione-S-transferase(GST)), a hemagglutinin (HA) tag (corresponding to an epitope derivedfrom the influenza hemagglutinin protein; Wilson et al., Cell 37:767(1984)), maltose binding protein sequences, the FLAG epitope utilized inthe FLAGS extension/affinity purification system (Immunex Corp, Seattle,Wash.) (commercially available FLAG epitopes also are available throughKodak (New Haven, Conn.)), an E-epitope tag (E-tag), thioredoxin (TRX),avidin, and the like. Purification-facilitating epitope tags have beendescribed in the art (see, e.g., Whitehorn et al., Biotechnology13:1215-19 (1995)). A polypeptide can include an e-his tag, which maycomprise a polyhistidine sequence and an anti-e-epitope sequence(Pharmacia Biotech Catalog); e-his tags can be made by standardtechniques. The inclusion of a protease-cleavable polypeptide linkersequence between the purification domain and the polypeptide is usefulto facilitate purification. The histidine residues facilitatepurification on IMIAC (immobilized metal ion affinity chromatography(IMAC), as described in Porath et al. Protein Expression andPurification 3:263-281 (1992)), while the enterokinase cleavage siteprovides a method for separating the polypeptide from the fusionprotein. pGEX vectors (Promega; Madison, Wis.) can also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption to ligand-agarosebeads (e.g., glutathione-agarose in the case of GST-fusion) followed byelution in the presence of free ligand. Additional examples ofpolypeptide purification facilitating subsequences and the use thereoffor protein purification are described in, e.g., Int'l Patent Appn Publ.No. WO 00/15823. After expression of the polypeptide of interest andisolation thereof by such fusion partners or otherwise as describedabove, protein refolding steps can be used, as desired, in completingconfiguration of the mature polypeptide.

A fusion protein of the invention also can include one or moreadditional peptide fragments or peptide portions which promote detectionof the fusion protein. For example, a reporter peptide fragment orportion (e.g., green fluorescent protein (GFP), β-galactosidase, or adetectable domain thereof) can be incorporated in the fusion protein.Additional marker molecules that can be conjugated to the polypeptide ofthe invention include radionuclides, enzymes, fluorophores, smallmolecule ligands, and the like. Such detection-promoting fusion partnersare particularly useful in fusion proteins used in diagnostic techniquesdiscussed elsewhere herein.

In another aspect, a polypeptide of the invention can comprise a fusionpartner that promotes stability of the polypeptide, secretion of thepolypeptide (other than by signal targeting), or both. For example, thepolypeptide can comprise an immunoglobulin (Ig) domain, such as an IgGpolypeptide comprising an Fc hinge region, a CH2 domain, and a CH3domain, that promotes stability and/or secretion of the polypeptide.

The fusion protein peptide fragments or peptide portions can beassociated in any suitable manner. The various polypeptide fragments orportions of the fusion protein may be covalently associated (e.g., bymeans of a peptide or disulfide bond). The polypeptide fragments orportions can be directly fused (e.g., the C-terminus of an antigenic orimmunogenic sequence of the invention can be fused to the N-terminus ofa purification sequence or heterologous immunogenic sequence). Thefusion protein can include any suitable number of modified bonds, e.g.,isosteres, within or between the peptide portions. Alternatively oradditionally, the fusion protein can include a peptide linker betweenone or more polypeptide fragments or portions that includes one or moreamino acid sequences not forming part of the biologically active peptideportions. Any suitable peptide linker can be used. Such a linker can beany suitable size. Typically, the linker is less than about 30 aminoacid residues, less than about 20 amino acid residues, and/or less than10 amino acid residues. The linker predominantly may comprise or consistof neutral amino acid residues. Suitable linkers are generally describedin, e.g., U.S. Pat. Nos. 5,990,275, 6,010,883, 6,197,946, and EuropeanPatent Application 0 035 384. If separation of peptide fragments orpeptide portions is desirable a linker that facilitates separation canbe used. An example of such a linker is described in U.S. Pat. No.4,719,326. “Flexible” linkers, which are typically composed ofcombinations of glycine and/or serine residues, can be advantageous.Examples of such linkers are described in, e.g., McCafferty et al.,Nature 348:552-554 (1990), Huston et al., Proc. Natl. Acad. Sci. USA85:5879-5883 (1988), Glockshuber et al., Biochemistry 29:1362-1367(1990), and Cheadle et al., Molecular Immunol. 29:21-30 (1992), Bird etal., Science 242:423-26 (1988), and U.S. Pat. Nos. 5,672,683, 6,165,476,and 6,132,992.

The use of a linker also can reduce undesired immune response to thefusion protein created by the fusion of the two peptide fragments orpeptide portions, which can result in an unintended MHC I and/or MHC IIepitope being present in the fusion protein. In addition to the use of alinker, identified undesirable epitope sequences or adjacent sequencescan be PEGylated (e.g., by insertion of lysine residues to promote PEGattachment) to shield identified epitopes from exposure. Othertechniques for reducing immunogenicity of the fusion protein of theinvention can be used in association with the administration of thefusion protein include the techniques provided in U.S. Pat. No.6,093,699.

Making Polypeptides

Recombinant methods for producing and isolating polypeptides of theinvention (including fusion proteins of the invention) are describedbelow. In addition to recombinant production, the polypeptides may beproduced by direct peptide synthesis using solid-phase techniques (see,e.g., Stewart et al. (1969) Solid-Phase Peptide Synthesis, W.H. FreemanCo, San Francisco; Merrifield (1963) J. Am. Chem. Soc 85:2149-2154).Peptide synthesis may be performed using manual techniques or byautomation. Automated synthesis may be achieved, for example, usingApplied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City,Calif.) in accordance with the instructions provided by themanufacturer. For example, subsequences may be chemically synthesizedseparately and combined using chemical methods to provide mutant CTLA-4polypeptides or functional fragments thereof. Alternatively, suchsequences may be ordered from any number of companies that specialize inproduction of polypeptides. Most commonly, polypeptides of the inventionare produced by expressing coding nucleic acids and recoveringpolypeptides, e.g., as described below.

The invention provides methods for producing polypeptides (includingfusion proteins) of the invention. One such method comprises introducinginto a population of cells any nucleic acid described herein, which isoperatively linked to a regulatory sequence effective to produce theencoded polypeptide, culturing the cells in a culture medium to producethe polypeptide, and isolating the polypeptide from the cells or fromthe culture medium. An amount of nucleic acid sufficient to facilitateuptake by the cells (transfection) and/or expression of the polypeptideis utilized. The culture medium can be any described herein and in theExamples. Additional media are known to those of skill in the art. Thenucleic acid is introduced into such cells by any delivery methoddescribed herein, including, e.g., injection, needleless injectiondevice, gene gun, electroporation (e.g., DNA electroporation device,Inovio Biomedical Corp. (San Diego)), transdermal delivery, passiveuptake, etc. The nucleic acid of the invention may be part of a vector,such as a recombinant expression vector, including a DNA plasmid vector,viral vector, or any vector described herein. The nucleic acid or vectorcomprising a nucleic acid of the invention may be prepared andformulated as described herein, above, and in the Examples below. Such anucleic acid or expression vector may be introduced into a population ofcells of a mammal in vivo, or selected cells of the mammal (e.g., tumorcells) may be removed from the mammal and the nucleic acid expressionvector introduced ex vivo into the population of such cells in an amountsufficient such that uptake and expression of the encoded polypeptideresults. Or, a nucleic acid or vector comprising a nucleic acid of theinvention is produced using cultured cells in vitro. In one aspect, themethod of producing a polypeptide of the invention comprises introducinginto a population of cells a recombinant expression vector comprisingany nucleic acid described herein in an amount and formula such thatuptake of the vector and expression of the polypeptide will result;administering the expression vector into a mammal by anyintroduction/delivery format described herein; and isolating thepolypeptide from the mammal or from a byproduct of the mammal. Suitablehost cells, expression vectors, methods for transfecting host cells withan expression vector comprising a nucleic acid sequence encoding apolypeptide of the invention, cell cultures, and procedures forproducing and recovering such polypeptide from a cell culture aredescribed in detail below in the section entitled “Nucleic Acids of theInvention.” Additional methods of production are discussed in theExamples, infra.

As noted above, polypeptides of the invention (which includes fusionproteins of the invention) can be subject to various changes, such asone or more amino acid or nucleic acid insertions, deletions, andsubstitutions, either conservative or non-conservative, including where,e.g., such changes might provide for certain advantages in their use,e.g., in their therapeutic or prophylactic use or administration ordiagnostic application. Procedures for making variants of polypeptidesby using amino acid substitutions, deletions, insertions, and additionsare routine in the art. Polypeptides and variants thereof having thedesired ability to bind CD80 and/or CD86, or a fragment thereof (e.g.,ECD) or an ability to suppress an immune response in vitro or in vivo asdescribed in detail elsewhere herein are readily identified by assaysknown to those of skill in the art and by the assays described herein.See, e.g., assays presented in the Examples below.

The nucleic acids of the invention, discussed in greater detail infra,can also be subject to various changes, such as one or moresubstitutions of one or more nucleic acids in one or more codons suchthat a particular codon encodes the same or a different amino acid,resulting in either a conservative or non-conservative substitution, orone or more deletions of one or more nucleic acids in the sequence. Thenucleic acids can also be modified to include one or more codons thatprovide for optimum expression in an expression system (e.g., mammaliancell or mammalian expression system), while, if desired, said one ormore codons still encode the same amino acid(s). Procedures for makingvariants of nucleic acids by using nucleic acid substitutions,deletions, insertions, and additions, and degenerate codons, are routinein the art, and nucleic acid variants encoding polypeptides having thedesired properties described herein (e.g., an ability to bind CD80and/or CD86, and/or suppress an immune response in vitro or in vivo) arereadily identified using the assays described herein. Such nucleic acidchanges might provide for certain advantages in their therapeutic orprophylactic use or administration, or diagnostic application. In oneaspect, the nucleic acids and polypeptides can be modified in a numberof ways so long as they comprise a nucleic acid or polypeptide sequencesubstantially identical to the nucleic acid sequence of a respectivemutant CTLA-4 polypeptide-encoding nucleic acid or mutant CTLA-4polypeptide of the invention, respectively.

Nucleic Acids of the Invention

The invention provides isolated or recombinant nucleic acids (alsoreferred to herein as polynucleotides), collectively referred to as“nucleic acids of the invention” (or “polynucleotides of theinvention”), which encode polypeptides of the invention. Nucleic acidsof the invention, including all described below, are useful inrecombinant production (e.g., expression) of polypeptides of theinvention, typically through expression of a plasmid expression vectorcomprising a sequence encoding the polypeptide or fragment thereof; astherapeutics; as prophylactics; as diagnostic tools; as diagnosticprobes for the presence of complementary or partially complementarynucleic acids (including for detection of a wild-type CTLA-4 nucleicacid). For example, nucleic acids of the invention, including alldescribed below, are useful because they encode polypeptides that areuseful in suppressing or inhibiting an immune response (e.g., T cellactivation, T cell proliferation, cytokine synthesis or production(e.g., production of TNF-α, IFN-γ, IL-2), induction of activationmarkers (e.g., CD25, IL-2 receptor), inflammation, anti-collagenantibody production, and/or T cell-dependent antibody response) in vitroand/or in vivo applications, including, e.g., prophylactic and/ortherapeutic methods for treating immune system diseases, disorders, andconditions in which suppression of an immune response is desirable(e.g., methods for treating autoimmune diseases and disorders andmethods for inhibiting rejection of a tissue, cell, or organ transplantfrom a donor by a recipient). Nucleic acids of the invention can also beincorporated into expression vectors useful for gene therapy, DNAvaccination, and immunosuppressive therapy. Additional uses of thenucleic acids and vectors of the invention comprising such nucleic acidsare described elsewhere herein.

In one aspect, the invention provides an isolated or recombinant nucleicacid comprising a nucleotide sequence encoding any polypeptide(including any fusion protein, etc.) of the invention described above inthe section entitled “Polypeptides of the Invention” and elsewhereherein. The invention also provides an isolated or recombinant nucleicacid comprising a nucleotide sequence encoding a combination of two ormore of any polypeptides (including any fusion proteins) of theinvention described above and elsewhere herein. Also included is anucleic acid that encodes any polypeptide of the invention, such as,e.g., a mutant CTLA-4 ECD polypeptide or mutant CTLA-4-Ig fusionprotein, which comprises a sequence of codons substantially optimizedfor expression in a mammalian host, such as a human.

For example, in one aspect, the invention provides an isolated orrecombinant nucleic acid comprising a polynucleotide sequence thatencodes a polypeptide comprising a polypeptide sequence that has atleast 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to at least onepolypeptide sequence selected from the group consisting of SEQ IDNOS:1-73, or a complementary polynucleotide sequence thereof, whereinthe polypeptide binds CD80 and/or CD86 or a polypeptide fragment of CD80and/or CD86 (e.g., an extracellular domain of CD80 and/or CD86), and/orsuppress an immune response in vitro and/or in vivo, or a complementarypolynucleotide sequence thereof. Additional details regarding thefunctional properties and characteristics of such polypeptides arediscussed above in “Polypeptides of the Invention.” Some such nucleicacids encode a polypeptide comprising a polypeptide sequence having anamino acid length about equal to the amino acid length of the hCTLA-4ECD; as, e.g., 110-138, 112-132, 118-130, 119-129, 120-128, 121-127,122-126, 123-125, or 124 amino acid residues. Exemplary nucleic acidswhich encode the mutant CTLA-4 ECD polypeptides comprising the sequencesset forth in SEQ ID NOS:1-73, but are not limited to, e.g., nucleicacids having nucleotide sequences set forth in SEQ ID NOS:80-152,respectively. For example, an exemplary nucleic acid encoding thepolypeptide shown in SEQ ID NO:1 (clone D3-1) is the nucleic acid shownin SEQ ID:80. Also included are fragments of any such nucleic acids,wherein such fragment encodes a polypeptide that binds CD80 and/or CD86and/or an ECD of either or both, and/or has an ability to suppress animmune response.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes apolypeptide (e.g., mutant CTLA-4 ECD polypeptide) which comprises apolypeptide sequence (a) which differs from a polypeptide sequenceselected from the group consisting of SEQ ID NOS:1-73 in no more than 6amino acid residues (e.g., no more than 1, 2, 3, 4, 5, or 6 amino acidresidues), and (b) wherein the amino acid residue in the polypeptidesequence at position 41, 50, 54, 55, 56, 64, 65, 70, or 85 is identicalto the amino acid residue at the corresponding position of saidpolypeptide sequence selected from the group consisting of SEQ IDNOS:1-73, wherein the polypeptide binds CD80 and/or CD86 and/or anextracellular domain of either or both, and/or inhibits an immuneresponse in vitro and/or in vivo, or a complementary polynucleotidesequence thereof. That is, the amino acid residue at such position 41,50, 54, 55, 56, 64, 65, 70, or 85 in such selected polypeptide sequenceis not deleted or substituted. Some such nucleic acids encodepolypeptides comprising a sequence which differs from the selectedpolypeptide sequence by no more than 6 amino acid residues and whichincludes amino acid residues at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,and/or 14 positions selected from amino acid positions 24, 30, 32, 41,50, 54, 55, 56, 64, 65, 70, 85, 104 and 106 that are identical to theamino acid residues at the corresponding positions in the selectedpolypeptide sequence. Such polypeptides can differ from the selectedpolypeptide sequence by amino acid deletion(s), addition(s), and/oramino acid substitution(s). An amino acid substitution may be aconservative or non-conservative substitution. Exemplary conservativesubstitutions are discussed in the section entitled “SequenceVariation.” Some such polypeptides comprise a sequence having a lengthof about 118-130, 119-129, 120-128, 121-127, 122-126, 123-125, or 124amino acid residues. Additional details of the functional properties andcharacteristics of such polypeptides are discussed above. Exemplarynucleic acids include, but are not limited to, e.g., those comprisingnucleotide sequences set forth in SEQ ID NOS:80-152.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes apolypeptide (e.g., mutant CTLA-4 ECD) comprising a polypeptide sequencewhich (a) differs from the polypeptide sequence of the extracellulardomain of human CTLA-4 shown in SEQ ID NO:159 in no more than 6 aminoacid residues, and (b) comprises at least one amino acid substitution atan amino acid position corresponding to position 50, 54, 55, 56, 64, 65,70, or 85 relative to the polypeptide sequence of SEQ ID NO:159, whereinthe polypeptide binds hCD80 and/or hCD86 and/or an ECD of either orboth, and/or inhibits an immune response in vitro and/or in vivo, or acomplementary polynucleotide sequence thereof. Some such nucleic acidsencode polypeptides comprising 2, 3, 4, 5, or 6 amino acid substitutionsat positions relative to the sequence set forth in SEQ ID NO:159selected from the group consisting of position 50, 54, 55, 56, 64, 65,70, and 85. Some such nucleic acids encoding polypeptides furthercomprising an amino acid substitution at a position corresponding toposition 104 and/or 30 relative to SEQ ID NO:159. Some such nucleicacids encode polypeptides comprising at least one amino acidsubstitution relative to SEQ ID NO:159 at position 70 (e.g., S70F),position 64 (e.g., S64P), position 50 (e.g., A50M), position 54 (e.g.,M54K/V), position 65 (e.g., I65S), position 56 (e.g., N56D), position 55(e.g., G55E), position 85 (e.g., M85A), and/or position 24 (e.g., A24E).Any such polypeptide may further comprise an amino acid substitutionrelative to SEQ ID NO:159 at position 104 (optionally L104E/D, e.g.,L104E), position 30 (e.g., T30N/D/A), and/or position 32 (e.g., V32I).Some such nucleic acids encode polypeptides comprising one or moresubstitutions at amino acid positions relative to SEQ ID NO:159 selectedfrom the group consisting of A50M, M54K, G55E, N56D, S64P, I65S, andS70F. Some such encoded polypeptides comprise a sequence having an aminoacid length of about 118-130, 119-129, 120-128, 121-127, 122-126,123-125, or 124 amino acid residues. Additional details of thefunctional properties and characteristics of such polypeptides arediscussed above.

The invention also provides an isolated or recombinant nucleic acidcomprising a polynucleotide sequence which encodes a polypeptide (e.g.,mutant CTLA-4 ECD) comprising a polypeptide sequence comprising (i) atleast 95%, 96%, 97%, 98%, 99%, or 100% identity to any polypeptidesequence selected from the group consisting of SEQ ID NOS:1-73 and (ii)a phenylalanine residue at an amino acid position corresponding toposition 70 of said polypeptide sequence selected from the groupconsisting of SEQ ID NO:1-73, wherein the polypeptide binds hCD80 and/orhCD86 or an ECD thereof and/or inhibits an immune response, or acomplementary polynucleotide sequence thereof. Some such nucleic acidsencode polypeptides comprising one or more of the following relative tothe selected sequence: a glutamic acid residue at an amino acid positioncorresponding to position 24; an asparagine residue at an amino acidposition corresponding to position 30; an isoleucine residue at an aminoacid position corresponding to position 32; a methionine residue at anamino acid position corresponding to position 50; a lysine residue at anamino acid position corresponding to position 54; a glutamic acidresidue at an amino acid position corresponding to position 55; anaspartic acid residue at an amino acid position corresponding toposition 56; a proline residue at an amino acid position correspondingto position 64; a serine residue at an amino acid position correspondingto position 65; and a glutamic acid residue at an amino acid positioncorresponding to position 104. Some such nucleic acids encodepolypeptides comprising a polypeptide sequence having a length of about118-130, 119-129, 120-128, 121-127, 122-126, 123-125, or 124 amino acidresidues. Additional details of the functional properties andcharacteristics of such polypeptides are discussed above.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes apolypeptide (e.g., mutant CTLA-4 ECD) comprising a polypeptide sequencewhich (a) differs from the polypeptide sequence of human CTLA-4 ECDpolypeptide shown in SEQ ID NO:159 in no more than 6 amino acidresidues, and (b) comprises at least one amino acid substitution,wherein said at least one amino acid substitution is S70F, wherein aminoacid residue positions are numbered according to SEQ ID NO:159, whereinthe polypeptide that binds hCD80 and/or hCD86 (and/or an ECD of eitheror both) and/or inhibits an immune response, or a complementarypolynucleotide sequence thereof. Some such nucleic acids encode apolypeptide that further comprises at least one amino acid substitutionselected from the group consisting of A24E, T30N, V32I, D41G, A50M,M54K, G55E, N56D, S64P, I65S, M85A, L104E, and I106F. Some such nucleicacids encode polypeptides comprising a polypeptide sequence having alength of about 118-130, 119-129, 120-128, 121-127, 122-126, 123-125, or124 amino acid residues. Additional details of the functional propertiesand characteristics of such polypeptides are discussed above.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes apolypeptide (e.g., mutant CTLA-4 ECD) comprising a polypeptide sequencewhich (a) differs from the polypeptide sequence shown in SEQ ID NO:31 inno more than 6 amino acid residues, and (b) comprises at least one ofthe following: a methionine residue at a position corresponding toposition 50 of SEQ ID NO:31, a lysine residue at a positioncorresponding to position 54 of SEQ ID NO:31, a glutamic acid residue ata position corresponding to position 55 of SEQ ID NO:31, a prolineresidue at a position corresponding to position 64 of SEQ ID NO:31, aserine residue at a position corresponding to position 65 of SEQ IDNO:31, a phenylalanine residue at a position corresponding to position70 of SEQ ID NO:31, wherein amino acid residue positions are numberedaccording to SEQ ID NO:31, and wherein the polypeptide binds CD80 and/orCD86 and/or an ECD of either or both, and/or inhibits an immuneresponse, or a complementary polynucleotide sequence thereof. Some suchencoded polypeptides comprise a glutamic acid residue at a positioncorresponding to position 104, an asparagine acid residue at a positioncorresponding to position 30, and/or an isoleucine residue at a positioncorresponding to position 32 of SEQ ID NO:31. Some such nucleic acidsencode polypeptides comprising a polypeptide sequence having a length ofabout 118-130, 119-129, 120-128, 121-127, 122-126, 123-125, or 124 aminoacid residues. Additional details of the functional properties andcharacteristics of such polypeptides are discussed above.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence having at least 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5%, or 100% identity to at least one polynucleotide sequenceselected from the group consisting of SEQ ID NOS:80-158, 201-204, 223,and 224, or a complementary polynucleotide sequence thereof, wherein thenucleic acid encodes a polypeptide that binds hCD80 and/or hCD86 and/oran ECD of either or both, and/or suppresses an immune response, or acomplementary polynucleotide sequence thereof. Exemplary nucleic acidscomprising polynucleotide sequences identified by SEQ ID NOS:80-158encode exemplary polypeptides comprising polypeptide sequencesidentified by SEQ ID NOS:1-79, respectively. Exemplary nucleic acidscomprising polynucleotide sequences identified by SEQ ID NOS:201-204encode exemplary polypeptides comprising polypeptide sequencesidentified by SEQ ID NOS:197-200, respectively.

In another aspect, the invention includes an isolated or recombinantnucleic acid comprising: (a) a polynucleotide sequence having at least95%, 96%, 97%, 98%, 99%, or 100% identity to an RNA polynucleotidesequence, wherein the RNA polynucleotide sequence comprises a DNAsequence selected from the group consisting of SEQ ID NOS:80-158,201-204, 223, and 224 in which all of the thymine nucleotide residues inthe DNA sequence are replaced with uracil nucleotide residues; (b) acomplementary polynucleotide sequence of (a); or (c) a fragment of anypolynucleotide sequence of (a) or (b), wherein the nucleic acid encodesa polypeptide that (i) binds CD80 and/or CD86 and/or an ECD of either orboth, and/or (ii) has an ability to suppress an immune response in vitroor in vivo (e.g., T cell activation or proliferation, cytokine synthesisor production (e.g., production of TNF-α, IFN-γ, IL-2), induction ofactivation markers (e.g., CD25, IL-2 receptor), inflammation,anti-collagen antibody production, and/or T cell-dependent antibodyresponse), or a polynucleotide sequence thereof.

The invention includes an isolated or recombinant nucleic acid encodingany multimer of any polypeptide of the invention described above (e.g.,dimer, tetramer, etc.). A discussed in greater detail elsewhere, a dimercomprising two polypeptides of the invention (including two fusionproteins) is typically formed during cellular processing by thegeneration of one or more covalent disulfide bonds between cysteineresidue(s) in one polypeptide and cysteine residue(s) in the secondpolypeptide. Other multimers may be similarly formed. For example, in anon-limiting aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes arecombinant polypeptide dimer comprising two polypeptides, wherein eachsuch polypeptide comprises a polypeptide sequence having at least 95%,96%, 97%, 98%, 99%, or 100% identity to a sequence selected from thegroup consisting of SEQ ID NOS:1-73, wherein the dimer binds hCD80and/or hCD86 and/or inhibits an immune response, or a complementarypolynucleotide sequence thereof. Also included is an isolated orrecombinant nucleic acid comprising a polynucleotide sequence whichencodes a polypeptide dimer comprising two polypeptides, wherein eachsuch polypeptide differs from the polypeptide sequence of the hCTLA-4ECD (SEQ ID NO:159) in no more than 6 amino acid residues and comprisesat least one substitution at an amino acid position relative to SEQ IDNO:159 selected from the group consisting of A50M, M54K, G55E, N56D,S64P, I65S, and S70F; and which polypeptide optionally further comprisesthe substitution L104E, wherein said dimer binds hCD80 and/or hCD86and/or inhibits an immune response, or a complementary polynucleotidesequence thereof. Additional details of the functional properties ofsuch dimers are discussed above.

The invention also provides an isolated or recombinant nucleic acidencoding any fusion protein of the invention, including any multimericfusion protein of the invention (e.g., dimers, tetramers, etc.). In oneaspect, the invention provides an isolated or recombinant nucleic acidcomprising a polynucleotide sequence which encodes a fusion proteincomprising (a) a polypeptide comprising a polypeptide sequence that hasat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, or 100% identity to at least one polypeptide sequence selectedfrom the group consisting of SEQ ID NOS:1-73, and (b) an Ig polypeptide,wherein the fusion protein binds CD80 and/or CD86 (and/or CD80-Ig and/orCD86-Ig), and/or has an ability to suppress an immune response, or acomplementary polynucleotide sequence thereof. The Ig polypeptide maycomprise an Ig Fc polypeptide, including, e.g., an Ig Fc polypeptidecomprising a polypeptide sequence having at least 95%, 96%, 97%, 98%,99%, or 100% identity to a polypeptide sequence selected from the groupconsisting of SEQ ID NOS:184-186 and 218. A dimeric fusion proteincomprising two such monomeric fusion proteins is typically formed duringcellular processing by the generation of covalent disulfide bondsbetween cysteine residues in one monomeric fusion protein and cysteineresidues in the second monomeric fusion protein. Other multimers may besimilarly formed. Additional details of the functional properties andcharacteristics of such fusion proteins are discussed above.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes aprotein dimer (e.g., mutant CTLA-4-Ig dimer) comprising two monomericfusion proteins (e.g., monomeric mutant CTLA-4 Ig), each monomericfusion protein comprising: (a) a polypeptide (e.g., mutant CTLA-4 ECD)comprising a polypeptide sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to at least onepolypeptide sequence selected from the group consisting of SEQ IDNOS:1-73, and (b) an Ig polypeptide, wherein the fusion protein dimerbinds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig), and/or has anability to inhibit or suppress an immune response, or a complementarypolynucleotide sequence thereof. The two monomeric fusion proteins, uponexpression, are linked together via at least one disulfide bond formedbetween two cysteine residues present in each monomeric mutant CTLA-4-Igfusion protein. The Ig polypeptide may comprise an Ig Fc polypeptide,including, e.g., an Ig Fc polypeptide comprising a sequence having atleast 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selectedfrom the group consisting of SEQ ID NOS:184-186 and 218. In someinstances, the C-terminus of the polypeptide of (a) is covalently linkedor fused to the N-terminus of the Ig Fc polypeptide of (b). Additionaldetails of the functional properties and characteristics of such dimersare discussed above.

A stop codon (e.g., tga) is typically included at the C-terminus of eachnucleic acid sequence when the sequence is included in an expressionvector for expression of a protein of interest. For example, each of thenucleotide sequences of the invention encoding a mutant CTLA-4polypeptide or mutant CTLA-4 fusion protein may optionally furtherinclude a stop codon at the C terminus, such as TAA. A different stopcodon may be substituted for the TAA, such as a TGA stop codon. Anucleic acid sequence encoding a wild-type fusion protein (e.g.,hCTLA-4-Ig, hCD86-Ig, etc.) may also include a stop codon at itsC-terminus. Each of the nucleotide sequences may optionally furtherincludes at the N-terminus a nucleotide sequence encoding a signalpeptide to facilitate secretion of a mutant CTLA-4 polypeptide or fusionprotein.

Exemplary mutant CTLA-4-Ig fusion protein dimers include thosecomprising polypeptide sequences shown in SEQ ID NOS:74-79, 197-200,205-214, and 219-222. Exemplary nucleic acids encoding the mutant CTLA-4Ig fusion proteins of SEQ ID NOS:74-79, 197-200, 220 and 222 are setforth in SEQ ID NO:153-158, 201-204, and 223-224, respectively. Thefusion protein sequences of SEQ ID NOS:205-210, 211-214, 219, and 221are identical to the protein sequences of SEQ ID NOS:74-79, 197-200,220, and 222, respectively, except that the protein sequences of SEQ IDNOS:205-210 do not include the C-terminal lysine (K) residue; asexplained above, it is believed that the predicted C-terminal lysineresidue, which is encoded by the AAA codon immediately preceding the TAAstop codon of each such polynucleotide sequence, is cleaved from theresulting fusion protein during processing or secretion. The nucleicacid sequences of SEQ ID NOS:153-158, 201-204, and 223-224 encode thefusion protein sequences of SEQ ID NOS:74-79, 197-200, 220, and 222,respectively, each of which after cleavage/loss of the C-terminal Kresidue results in the fusion protein sequences shown in SEQ IDNOS:205-210, 211-214, 219, and 221, respectively.

Each of the polynucleotide sequences SEQ ID NOS:153-158, 201-204, and223-224 also includes at its N-terminus a nucleotide sequence encodingthe signal peptide shown in SEQ ID NO:181 or 215, which signal peptideis ultimately cleaved to form the mature fusion protein. Nucleotideresidues 1-111 of each of the polynucleotide sequences of SEQ IDNOS:153-158, 201-204, and 223-224, as counted from the N-terminus ofeach such polynucleotide sequence (nucleotide residues 1-111 are setforth in SEQ ID NO:215), encode the 37-amino acid residue WT hCTLA-4signal peptide shown in SEQ ID NO:216, which signal peptide isultimately cleaved upon expression of the mature mutant CTLA-4 fusionprotein monomer or dimer; thus, for each of the nucleic acid sequencesof SEQ ID NOS:153-158, 201-204, and 223-224, the first codon encodingthe first amino acid residue (methionine) of the mature IgG2 fusionprotein is composed of nucleotide residues 112-114 of said nucleotidesequence. As noted above, in some instances, the signal peptide sequencemay comprise only amino acid residues 1-35 as shown in SEQ ID NO:182 andthe nucleotide sequence encoding this 35-amino acid residue signalpeptide is shown in SEQ ID NO:181. Nevertheless, the encoded lysine (K)and alanine (A) residues at positions 36 and 37, respectively (encodedby the two codons AAA-GCC), are not present in the resulting maturemutant CTLA-4-Ig fusion protein and are believed cleaved from the maturemutant CTLA-4-Ig fusion protein during processing. The mature mutantCTLA-4 protein sequence typically begins with the methionine residuepresent at amino acid residue position 38 of the encoded mutantCTLA-4-Ig fusion protein.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes a fusionprotein dimer (e.g., mutant CTLA-4-Ig dimer) comprising two identicalmonomeric fusion proteins (e.g., monomeric mutant CTLA-4-Ig), whereineach such monomeric fusion protein comprises a polypeptide sequencehaving at least 95%, 96%, 97%, 98%, 99% or 100% identity to apolypeptide sequence selected from the group consisting of SEQ IDNOS:74-79, 197-200, 205-214, and 219-222, wherein the fusion proteindimer binds CD80 and/or CD86 (and/or a CD80-Ig and/or CD86-Ig), and/orhas an ability to inhibit an immune response, or a complementarypolynucleotide sequence thereof. Also provided is an isolated orrecombinant nucleic acid encoding such monomeric fusion protein whichbinds CD80 and/or CD86 (and/or a CD80-Ig and/or CD86-Ig, and/or has anability to inhibit an immune response. Exemplary mutant CTLA-4-Ig fusionprotein dimers include those comprising polypeptide sequences shown inSEQ ID NOS:74-79, 197-200, 220, and 222; exemplary nucleic acidsencoding such mutant CTLA-4 Ig fusion proteins, which are expressed asmutant CTLA-4-Ig fusion protein dimers, include those comprisingpolynucleotide sequences shown in SEQ ID NOS:153-158, 201-204, and223-224, respectively. Additional exemplary mutant CTLA-4-Ig fusionprotein dimers comprise the polypeptide sequences of SEQ ID NOS:205-210,211-214, 219, and 222, which are expressed as fusion protein dimers;these fusion proteins lack the C-terminal lysine residue because it istypically cleaved during processing or prior to secretion. Exemplarynucleic acids encoding these fusion protein sequences with theC-terminal lysine (the lysine is cleaved subsequently) include thepolynucleotide sequences of SEQ ID NOS:153-158, 201-204, and 223-224,respectively.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes a fusionprotein, wherein said fusion protein comprises a polypeptide sequencehaving at least 95%, 96%, 97%, 98%, 99% or 100% identity to apolypeptide sequence selected from the group consisting of SEQ IDNOS:74-79, 197-200, 205-214, and 219-222, wherein the fusion proteinbinds CD80 and/or CD86 (and/or a CD80-Ig and/or CD86-Ig), and/or has anability to inhibit an immune response, or a complementary polynucleotidesequence thereof.

In another aspect, the invention includes an isolated or recombinantnucleic acid comprising a nucleotide sequence having at least 95%, 96%,97%, 98%, 99% or 100% sequence identity to a polynucleotide sequenceselected from the group consisting of SEQ ID NOS:153-158, 201-204, and223-224, wherein such nucleic acid encodes a mutant CTLA-4-Ig proteindimer binds CD80 and/or CD86 (and/or a CD80-Ig and/or CD86-Ig), and/orhas an ability to inhibit an immune response, or a complementarypolynucleotide sequence thereof.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes a fusionprotein dimer (e.g., mutant CTLA-4-Ig dimer) comprising two monomericfusion proteins (e.g., monomeric mutant CTLA-4-Ig), wherein each suchmonomeric fusion protein comprises: (1) a polypeptide (e.g., mutantCTLA-4 ECD) which comprises a polypeptide sequence which differs from apolypeptide sequence selected from the group consisting of SEQ IDNOS:1-73 in no more than 6 amino acid residues, and wherein the aminoacid residue in the polypeptide sequence at position 41, 50, 54, 55, 56,64, 65, 70, or 85 is identical to the amino acid residue at thecorresponding position of said selected polypeptide sequence (e.g., apolypeptide selected from SEQ ID NOS:1-73), and (2) an Ig Fcpolypeptide, wherein the dimer binds CD80 and/or CD86 (and/or CD80-Igand/or CD86-Ig), and/or inhibits an immune response, or a complementarypolynucleotide sequence thereof. Additional details of the functionalproperties and characteristics of such dimers are discussed above. Alsoprovided is a recombinant or isolated nucleic acid comprising anucleotide sequence which encodes such a monomeric fusion protein whichbinds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or has anability to inhibit an immune response.

In another aspect, the invention provides an isolated or recombinantnucleic acid comprising a polynucleotide sequence which encodes a fusionprotein dimer (e.g., mutant CTLA-4-Ig dimer) comprising two monomericfusion proteins (e.g., two monomeric mutant CTLA-4-Ig), wherein eachsuch monomeric fusion protein comprises: (1) a mutant CTLA-4extracellular domain polypeptide comprising a polypeptide sequence which(a) differs from a polypeptide sequence selected from the groupconsisting of SEQ ID NOS:1-73 in no more than 6 amino acid residues, and(b) comprises at least one amino acid substitution at an amino acidposition corresponding to position 50, 54, 55, 56, 64, 65, 70, or 85relative to the polypeptide sequence of SEQ ID NO:159; and (2) an Igpolypeptide, wherein the fusion protein dimer binds CD80 and/or CD86(and/or CD80-Ig and/or CD86-Ig), and/or inhibits an immune response, ora complementary polynucleotide sequence thereof. Additional details ofthe functional properties and characteristics of such dimers arediscussed above. The Ig polypeptide may comprise an Ig Fc polypeptide,including, e.g., an Ig Fc polypeptide comprising a sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identityto a sequence selected from the group consisting of SEQ ID NOS:184-186and 218. Some such polypeptides of (a) comprise at least onesubstitution at an amino acid position relative to SEQ ID NO:159selected from the group consisting of A50M, M54K, G55E, N56D, S64P,I65S, and S70F. Some such polypeptides of (a) further comprise an aminoacid substitution relative to SEQ ID NO:159 at position 104 (e.g.,L104E/D), position 30 (e.g., T30N/D/A), and/or position 32 (e.g., V32I).Also provided is a recombinant or isolated nucleic acid encoding suchmonomeric fusion protein which binds CD80 and/or CD86 (and/or CD80-Igand/or CD86-Ig) and/or has an ability to inhibit an immune response.

In another aspect, the invention includes an isolated or recombinantnucleic acid encoding a fusion protein dimer (e.g., mutant CTLA-4-Igdimer) comprising two monomeric fusion proteins (e.g., monomeric mutantCTLA-4-Ig), wherein each such monomeric fusion protein comprises: (1) apolypeptide (e.g., mutant CTLA-4 ECD) comprising a polypeptide sequencewhich (i) has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity to a polypeptide sequence selected from the group consisting ofSEQ ID NOS:1-73 and (ii) includes a phenylalanine residue at an aminoacid position corresponding to position 70 of said polypeptide sequenceselected from the group consisting of SEQ ID NO:1-73; and (2) an Igpolypeptide, wherein the fusion protein dimer binds CD80 and/or CD86(and/or CD80-Ig and/or CD86-Ig), and/or has an ability to inhibit animmune response, or a complementary polynucleotide sequence thereof. Theencoded Ig polypeptide may comprise an Ig Fc polypeptide comprising apolypeptide sequence having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identity to a polypeptide sequence selected from the groupconsisting of SEQ ID NOS:184-186 and 218. Some such encoded dimerscomprise one or more of the following relative to said selectedpolypeptide sequence of (1) (i): a Glu residue at a positioncorresponding to position 24; an Asn at a position corresponding toposition 30; an Ile residue at a position corresponding to position 32;a Met residue at a position corresponding to position 50; a Lys residueat a position corresponding to position 54; a Glu residue at a positioncorresponding to position 55; an Asp residue at a position correspondingto position 56; a Pro residue at a position corresponding to position64; a Ser residue at an amino acid position corresponding to position65; and a Glu residue at a position corresponding to position 104.Additional details of the functional properties and characteristics ofsuch dimers are discussed above. Also provided is a recombinant orisolated nucleic acid comprising a nucleotide sequence which encodessuch a monomeric fusion protein which binds CD80 and/or CD86 (and/orCD80-Ig and/or CD86-Ig) and/or has an ability to inhibit an immuneresponse.

In another aspect, the invention includes an isolated or recombinantnucleic acid encoding fusion protein dimer (e.g., mutant CTLA-4-Igdimer) comprising two monomeric fusion proteins (e.g., monomeric mutantCTLA-4-Ig), wherein each such monomeric fusion protein comprises: (1) apolypeptide (e.g., mutant CTLA-4 ECD) comprising a polypeptide sequencewhich (a) differs from the polypeptide sequence of the human CTLA-4 ECDpolypeptide shown in SEQ ID NO:159 in no more than 6 amino acidresidues, and (b) comprises at least one amino acid substitution,wherein said at least amino acid substitution comprises S70F, whereinamino acid residue positions are numbered according to SEQ ID NO:159;and (2) an IgG Fc polypeptide, wherein said dimer binds hCD80 and/orhCD86 (and/or hCD86-Ig and/or hCD86-Ig), and/or inhibits an immuneresponse, or a complementary polynucleotide sequence thereof. The Ig Fcpolypeptide may comprise a sequence having at least 95%, 96%, 97%, 98%,99%, or 100% identity to a polypeptide sequence selected from the groupconsisting of SEQ ID NOS:184-186 and 218. The encoded polypeptide of (1)may further comprise at least one amino acid substitution selected fromthe group consisting of A24E, T30N, V32I, D41G, A50M, M54K, G55E, N56D,S64P, I65S, M85A, L104E, and I106F. Additional details of the functionalproperties and characteristics of such dimers are discussed above. Alsoprovided is a recombinant or isolated nucleic acid comprising anucleotide sequence which encodes such a monomeric fusion protein whichbinds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or has anability to inhibit an immune response.

In another aspect, the invention provides an isolated or recombinantnucleic acid encoding fusion protein dimer (e.g., mutant CTLA-4-Igdimer) comprising two monomeric fusion proteins (e.g., monomeric mutantCTLA-4-Ig), wherein each such monomeric fusion protein comprises: (1) apolypeptide (e.g., mutant CTLA-4 ECD) comprising a polypeptide sequencewhich (a) differs from the polypeptide sequence of SEQ ID NO:31 in nomore than 6 amino acid residues, and (b) comprises at least one of thefollowing: a methionine residue at a position corresponding to position50 of SEQ ID NO:31, a lysine residue at a position corresponding toposition 54 of SEQ ID NO:31, a glutamic acid residue at a positioncorresponding to position 55 of SEQ ID NO:31, a proline residue at aposition corresponding to position 64 of SEQ ID NO:31, a serine residueat a position corresponding to position 65 of SEQ ID NO:31, aphenylalanine residue at a position corresponding to position 70 of SEQID NO:31, wherein amino acid residue positions are numbered according toSEQ ID NO:31; and (2) an Ig polypeptide, wherein said dimer binds hCD80and/or hCD86 (and/or hCD86-Ig and/or hCD86-Ig), and/or inhibits animmune response, or a complementary polynucleotide sequence thereof. TheIg polypeptide may comprise an Ig Fc polypeptide, including, e.g., an IgFc polypeptide comprising a sequence having at least 95%, 96%, 97%, 98%,99%, or 100% identity to a sequence selected from the group consistingof SEQ ID NOS:184-186 and 218. In some such dimers or monomers, thepolypeptide of (1) comprises a glutamic acid residue at a positioncorresponding to position 104, an asparagine acid residue at a positioncorresponding to position 30, and/or an isoleucine residue at a positioncorresponding to position 32 of SEQ ID NO:31. Additional details of thefunctional properties and characteristics of such dimers are discussedabove. Also provided is a recombinant or isolated nucleic acidcomprising a nucleotide sequence which encodes such a monomeric fusionprotein which binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig)and/or has an ability to inhibit an immune response.

Also included in the invention are fragments of any such nucleic acidsof the invention described above, wherein such fragments encode apolypeptide that binds hCD80 and/or hCD86 and/or an ECD of either orboth, and/or has an ability to suppress or inhibit an immune response.Many fragments of these nucleic acids will express polypeptides thatbind hCD80 and/hCD86 or an ECD thereof, or suppress an immune response,which properties can be readily identified with reasonableexperimentation. Nucleotide fragments typically comprise at least 250,300, 400, 500, 600, 700, 800, 900, 950, 1000 or more nucleotide bases.

The invention includes an isolated or recombinant nucleic acid thatencodes a protein comprising a signal peptide and a polypeptide of theinvention (which includes a dimeric or monomeric fusion protein of theinvention), such as a mutant CTLA-4 ECD polypeptide or mutant CTLA-4-Igfusion protein of the invention, that binds CD80 and/or CD86 and/orsuppresses an immune response in in vitro or in vivo assays and/ormethods as described in detail elsewhere herein. The encoded signalpeptide sequence, which directs secretion of the mature polypeptidethrough a prokaryotic or eukaryotic cell membrane, is typicallycovalently linked to the amino terminus of said polypeptide. A varietyof signal peptides can be used, including, e.g., the signal peptidesequence set forth in SEQ ID NO:182, which is encoded by, e.g., thenucleotide sequence shown in SEQ ID NO:181, or the signal peptidesequence set forth in SEQ ID NO:216, which is encoded by, e.g., thenucleotide sequence shown in SEQ ID NO:215. The invention also includean isolated or recombinant nucleic acid which encodes a proteincomprising a signal peptide, mutant CTLA-4 ECD polypeptide,transmembrane domain, and/or cytoplasmic domain as discussed detailabove.

The signal peptide sequence of the full-length human CTLA-4 protein canbe used to direct expression or secretion of a recombinant mutant CTLA-4ECD polypeptide or mutant CTLA-4-Ig fusion protein of the invention. Inone aspect, the signal peptide (SP) of the hCTLA-4 protein comprisesamino acid residues 1-37 of the hCTLA-4 protein; this signal peptidesequence is shown in SEQ ID NO:216. In this instance, the mature hCTLA-4protein typically begins with the methionine residue at position 38, andthe amino acid residues of the mature hCTLA-4 protein are numberedaccordingly beginning with this methionine residue being designated asthe first amino acid (i.e., occupying position 1). Thus, a signalpeptide comprising the peptide sequence shown in SEQ ID NO:216 can befused or linked to the amino (N) terminus of a mutant CTLA-4 ECDpolypeptide or mutant CTLA-4-Ig fusion protein of the invention, such asby a covalent linkage, so as to facilitate expression or secretion ofthe mutant CTLA-4 ECD polypeptide or mutant CTLA-4-Ig fusion protein,respectively. An exemplary nucleic acid comprising a nucleotide sequencethat encodes the hCTLA-4 signal peptide sequence of SEQ ID NO:216 is setforth in SEQ ID NO:215.

When the signal peptide sequence of SEQ ID NO:216 is fused to linked tothe N-terminus of a mutant CTLA-4 ECD polypeptide or mutant CTLA-4-Igfusion protein, upon expression or secretion of said polypeptide orfusion protein, the signal peptide is cleaved; the resulting maturemutant CTLA-4 ECD polypeptide or mature mutant CTLA-4-Ig fusion proteintypically begins with the methionine residue at position 38, and theamino acid residues of the mature mutant CTLA-4 ECD polypeptide ormature mutant CTLA-4-Ig fusion protein are numbered accordinglybeginning with this methionine residue being designated as the firstamino acid (i.e., occupying position 1).

The invention includes an isolated or recombinant polypeptide comprisinga signal peptide (e.g., SEQ ID NO:216) and a mutant CTLA-4 ECDpolypeptide (e.g., a sequence selected from the group of SEQ IDNOS:1-73), wherein the signal peptide is covalently linked to theN-terminus of the mutant CTLA-4 ECD polypeptide. Also included is anisolated or recombinant polypeptide comprising a signal peptide (e.g.,SEQ ID NO:216) and a mutant CTLA-4-Ig (e.g., a sequence selected fromthe group of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222), whereinthe signal peptide is covalently linked to the N-terminus of the mutantCTLA-4-Ig. Also provided is an isolated or recombinant nucleic acidcomprising a nucleotide sequence (e.g., SEQ ID NO:215) encoding a signalpeptide (e.g., SEQ NO:216) and a nucleotide sequence encoding a mutantCTLA-4 ECD polypeptide (e.g., a sequence selected from the group of SEQID NOS:1-73) or a mutant CTLA-4-Ig fusion protein (e.g., a sequenceselected from the group of SEQ ID NOS:74-79, 197-200, 205-214, and219-222).

In an alternative aspect, the signal peptide of the full-length hCTLA-4protein comprises residues 1-35 of the full-length hCTLA-4 protein; thissignal peptide comprises the peptide sequence shown in SEQ ID NO:182. Inthis instance, the two amino acid residues lysine (K) and alanine (A) atpositions 36 and 37, respectively, of the hCTLA-4 protein arenevertheless typically absent from the mature secreted hCTLA-4 proteinas determined by protein sequencing. Thus, the resulting mature hCTLA-4protein similarly begins with the methionine residue at position 38, andthe amino acid residues of the mature hCTLA-4 protein are numberedaccordingly beginning with this methionine residue at position 38 of thehCTLA-4 protein being designated as the first amino acid of the maturehCTLA-4 protein. Because amino acid residues lysine (K) and alanine (A)at positions 36 and 37, respectively, of the full-length hCTLA-4 proteinare not present in the resulting mature hCTLA-4 protein, it is believedthey have been cleaved from the mature hCTLA-4 protein duringprocessing. An exemplary nucleic acid comprising a nucleotide sequencethat encodes the hCTLA-4 signal peptide sequence (SEQ ID NO:182) isshown in SEQ ID NO:181.

A signal peptide comprising the peptide sequence shown in SEQ ID NO:182can be fused or linked to the N-terminus of a mutant CTLA-4 ECDpolypeptide or mutant CTLA-4-Ig fusion protein of the invention, such asby a covalent linkage, so as to facilitate expression or secretion ofthe mutant CTLA-4 ECD polypeptide or mutant CTLA-4-Ig fusion protein,respectively. When the signal peptide sequence of SEQ ID NO:182 is fusedor linked to the N-terminus of a mutant CTLA-4 ECD polypeptide or mutantCTLA-4-Ig fusion protein, upon expression or secretion of thepolypeptide or fusion protein, the signal peptide is cleaved; theresulting mature mutant CTLA-4 ECD polypeptide or mature mutantCTLA-4-Ig fusion protein nevertheless typically begins with themethionine residue at position 38, and the amino acid residues of themature mutant CTLA-4 ECD polypeptide or mature mutant CTLA-4-Ig fusionprotein are numbered accordingly beginning with this methionine residuebeing designated as the first amino acid (i.e., occupying position 1).Because amino acid residues lysine (K) and alanine (A) at positions 36and 37, respectively, of the full-length hCTLA-4 protein are not presentin the resulting mature mutant CTLA-4 ECD polypeptide or mature mutantCTLA-4-Ig fusion protein, it is believed they have been cleaved fromsaid mutant CTLA-4 ECD polypeptide or mutant CTLA-4-Ig fusion proteinduring processing.

The invention includes an isolated or recombinant polypeptide comprisinga signal peptide (e.g., SEQ ID NO:182) and a mutant CTLA-4 ECDpolypeptide (e.g., a sequence selected from the group of SEQ IDNOS:1-73), wherein the signal peptide is covalently linked to theN-terminus of the mutant CTLA-4 ECD polypeptide. Also included is anisolated or recombinant polypeptide comprising a signal peptide (e.g.,SEQ ID NO:182) and a mutant CTLA-4-Ig (e.g., a sequence selected fromthe group of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222), whereinthe signal peptide is covalently linked to the N-terminus of the mutantCTLA-4-Ig. Also provided is an isolated or recombinant nucleic acidcomprising a nucleotide sequence (e.g., SEQ ID NO:181) encoding a signalpeptide (e.g., SEQ NO: 182) and a nucleotide sequence encoding a mutantCTLA-4 ECD polypeptide (e.g., a sequence selected from the group of SEQID NOS:1-73) or a mutant CTLA-4-Ig fusion protein (e.g., a sequenceselected from the group of SEQ ID NOS:74-79, 197-200, 205-214, and219-222).

A nucleic acid of the invention can further comprise one or moresuitable additional nucleotide sequences. For example, given that apolypeptide of the invention (which includes a fusion protein of theinvention) can comprise one or more additional polypeptide sequences,such as, e.g., a polypeptide purification subsequence (such as, e.g., asubsequence is selected from an epitope tag, a FLAG tag, a polyhistidinesequence, and a GST fusion), signal peptide sequence, etc., theinvention includes nucleic acids that encode all such polypeptidescomprising such additional sequences. Exemplary signal peptides, whichupon expression are typically covalently linked to the N-terminal of apolypeptide of the invention, are discussed above. For example, anucleic acid encoding a polypeptide sequence of any of SEQ ID NOS:1-79,197-200, 205-214, and 219-222 can further comprise a nucleic acidencoding a signal peptide, such as the signal peptide sequence of SEQ IDNO:182, such as, e.g., the nucleotide sequence set forth in SEQ IDNO:181, or the signal peptide sequence set forth in SEQ ID NO:216, whichis encoded by, e.g., the nucleotide sequence shown in SEQ ID NO:215.Such nucleotide sequences can be directly fused together, in appropriatereading frame, such that the resulting nucleic acid comprises anucleotide sequence encoding a signal peptide of the invention and anucleotide sequence encoding a polypeptide of the invention.

A nucleic acid of the invention can be isolated by any suitabletechnique, of which several are known in the art. An isolated nucleicacid of the invention (e.g., a nucleic acid that is prepared in a hostcell and subsequently substantially purified by any suitable nucleicacid purification technique) can be re-introduced into a host cell orre-introduced into a cellular or other biological environment orcomposition wherein it is no longer the dominant nucleic acid speciesand is no longer separated from other nucleic acids.

Nearly any isolated or recombinant nucleic acid of the invention can beinserted in or fused to a suitable larger nucleic acid molecule(including e.g., but not limited to, a chromosome, a plasmid, anexpression vector or cassette, a viral genome, a gene sequence, a linearexpression element, a bacterial genome, a plant genome, or an artificialchromosome, such as a mammalian artificial chromosome (MAC), or theyeast and bacterial counterparts thereof (i.e., a YAC or a BAC) to forma recombinant nucleic acid using standard techniques. As anotherexample, an isolated nucleic acid of the invention can be fused tosmaller nucleotide sequences, such as promoter sequences,immunostimulatory sequences, and/or sequences encoding other aminoacids, such as other antigen epitopes and/or linker sequences to form arecombinant nucleic acid.

In some instances, a recombinant or synthetic nucleic acid may begenerated by chemical synthesis techniques applied outside of thecontext of a host cell (e.g., a nucleic acid produced through polymerasechain reaction (PCR) or chemical synthesis techniques, examples of whichare described further herein).

Nucleic acids encoding polypeptides (including fusion proteins) of theinvention can have any suitable chemical composition that permits theexpression of a polypeptide of the invention or other desired biologicalactivity (e.g., hybridization with other nucleic acids). Thepolynucleotides of the invention can be in the form of RNA or in theform of DNA, and include mRNA, cRNA, recombinant or synthetic RNA andDNA, and cDNA. The nucleic acids of the invention are typically DNAmolecules, and usually double-stranded DNA molecules. However,single-stranded DNA, single-stranded RNA, double-stranded RNA, andhybrid DNA/RNA nucleic acids or combinations thereof comprising any ofthe nucleotide sequences of the invention also are provided. A nucleicacid of the invention can include any suitable nucleotide base, baseanalog, and/or backbone (e.g., a backbone formed by, or including, aphosphothioate, rather than phosphodiester, linkage, e.g., DNAcomprising a phosphothioate or phosphorothioate backbone). A nucleicacid of the invention, if single-stranded, can be the coding strand orthe non-coding (i.e., antisense or complementary) strand. In addition toa nucleotide sequence encoding a polypeptide of the invention (e.g.,nucleotide sequence that comprise the coding sequence of a mutant CTLA-4ECD polypeptide or mutant CTLA-4-Ig), the polynucleotide of theinvention can comprise one or more additional coding nucleotidesequences, so as to encode, e.g., a fusion protein, targeting sequence(other than a signal sequence), or the like (more particular examples ofwhich are discussed further herein), and/or can comprise non-codingnucleotide sequences, such as introns, terminator sequence, or 5′ and/or3′ untranslated regions, which regions can be effective for expressionof the coding sequence in a suitable host, and/or control elements, suchas a promoter (e.g., naturally occurring or recombinant or shuffledpromoter).

Modifications to a nucleic acid are particularly tolerable in the 3rdposition of an mRNA codon sequence encoding such a polypeptide. Inparticular aspects, at least a portion of the nucleic acid comprises aphosphorothioate backbone, incorporating at least one syntheticnucleotide analog in place of or in addition to the naturally occurringnucleotides in the nucleic acid sequence. Also or alternatively, thenucleic acid can comprise the addition of bases other than guanine,adenine, uracil, thymine, and cytosine. Such modifications can beassociated with longer half-life, and thus can be desirable in nucleicacids vectors of the invention. Thus, in one aspect, the inventionprovides recombinant nucleic acids and nucleic acid vectors (discussedfurther below), which nucleic acids or vectors comprise at least one ofthe aforementioned modifications, or any suitable combination thereof,wherein the nucleic acid persists longer in a mammalian host than asubstantially identical nucleic acid without such a modification ormodifications. Examples of modified and/or non-cytosine, non-adenine,non-guanine, non-thymine nucleotides that can be incorporated in anucleotide sequence of the invention are provided in, e.g., the MANUALOF PATENT EXAMINING PROCEDURE §2422 (7th Revision—2000).

It is to be understood that a nucleic acid encoding at least one of thepolypeptides of the invention (which includes a fusion protein of theinvention), including those described above and elsewhere herein, is notlimited to a sequence that directly codes for expression or productionof a polypeptide of the invention. For example, the nucleic acid cancomprise a nucleotide sequence which results in a polypeptide of theinvention through intein-like expression (as described in, e.g., Colsonand Davis (1994) Mol. Microbiol. 12(3):959-63, Duan et al. (1997) Cell89(4):555-64, Perler (1998) Cell 92(1):1-4, Evans et al. (1999)Biopolymers 51(5):333-42, and de Grey, Trends Biotechnol. 18(9):394-99(2000)), or a nucleotide sequence which comprises self-splicing introns(or other self-spliced RNA transcripts), which form an intermediaterecombinant polypeptide-encoding sequence (as described in, e.g., U.S.Pat. No. 6,010,884). The nucleic acid also or alternatively can comprisesequences which result in other splice modifications at the RNA level toproduce an mRNA transcript encoding the polypeptide and/or at the DNAlevel by way of trans-splicing mechanisms prior to transcription(principles related to such mechanisms are described in, e.g., Chabot,Trends Genet. (1996) 12(11):472-78, Cooper (1997) Am. J. Hum. Genet.61(2):259-66, and Hertel et al. (1997) Curr. Opin. Cell. Biol.9(3):350-57). Due to the inherent degeneracy of the genetic code,several nucleic acids can code for any particular polypeptide of theinvention. Thus, for example, any of the particular nucleic acidsdescribed herein can be modified by replacement of one or more codonswith an equivalent codon (with respect to the amino acid called for bythe codon) based on genetic code degeneracy. Other nucleotide sequencesthat encode a polypeptide having the same or a functionally equivalentsequence as a polypeptide sequence of the invention can also be used tosynthesize, clone and express such polypeptide.

In general, any of the nucleic acids of the invention can be modified toincrease expression in a particular host, using the techniquesexemplified herein with respect to the above-described nucleic acidsencoding a polypeptide of the invention (e.g., nucleic acids encodingmutant CTLA-4 ECD or mutant CTLA-4-Ig). Any of the nucleic acids of theinvention as described herein may be codon optimized for expression in aparticular mammal (normally humans). A variety of techniques for codonoptimization are known in the art. Codons that are utilized most oftenin a particular host are called optimal codons, and those not utilizedvery often are classified as rare or low-usage codons (see, e.g., Zhang,S. P. et al. (1991) Gene 105:61-72). Codons can be substituted toreflect the preferred codon usage of the host, a process called “codonoptimization” or “controlling for species codon bias.” Optimized codingsequence comprising codons preferred by a particular prokaryotic oreukaryotic host can be used to increase the rate of translation or toproduce recombinant RNA transcripts having desirable properties, such asa longer half-life, as compared with transcripts produced from anon-optimized sequence. Techniques for producing codon-optimizedsequences are known (see, e.g., E. et al. (1989) Nuc. Acids Res.17:477-508). Translation stop codons can also be modified to reflecthost preference. For example, preferred stop codons for S. cerevisiaeand mammals are UAA and UGA respectively. The preferred stop codon formonocotyledonous plants is UGA, whereas insects and E. coli prefer touse UAA as the stop codon (see, e.g., Dalphin, M. E. et al. (1996) Nuc.Acids Res. 24:216-218). The arrangement of codons in context to othercodons also can influence biological properties of a nucleic acidsequences, and modifications of nucleic acids to provide a codon contextarrangement common for a particular host also is contemplated by theinventors. Thus, a nucleic acid sequence of the invention can comprise acodon optimized nucleotide sequence, i.e., codon frequency optimizedand/or codon pair (i.e., codon context) optimized for a particularspecies (e.g., the polypeptide can be expressed from a polynucleotidesequence optimized for expression in humans by replacement of “rare”human codons based on codon frequency, or codon context, such as byusing techniques such as those described in Buckingham et al. (1994)Biochimie 76(5):351-54 and U.S. Pat. Nos. 5,082,767, 5,786,464, and6,114,148).

Nucleic acids of the invention can be modified by truncation or one ormore residues of the C-terminus portion of the sequence. Additional, avariety of stop or termination codons may be included at the end of thenucleotide sequence as further discussed below.

One or more nucleic acids of the invention may be included in a vector,cell, or host environment in which a coding nucleotide sequence of theinvention is a heterologous gene.

Polynucleotides of the invention include polynucleotide sequences thatencode any polypeptide of the invention (or polypeptide fragmentthereof) which binds CD80 and/or CD86 and/or suppresses an immuneresponse, polynucleotides that hybridize under at least stringentconditions to one or more such polynucleotide sequences describedherein, polynucleotide sequences complementary to any suchpolynucleotide sequences, and variants, analogs, and homologuederivatives of all of the above. A coding sequence refers to anucleotide sequence encodes a particular polypeptide or a domain,subsequence, region, or fragment of said polypeptide. A coding sequencemay code for a mutant CTLA-4 polypeptide or fragment thereof having afunctional property, such as the ability to bind CD80 and/or CD86 and/orinhibit or suppress an immune response. A nucleic acid of the inventionmay comprise a respective coding sequence of a mutant CTLA-4 polypeptideof the invention, and variants, analogs, and homologue derivativesthereof.

Nucleic acids of the invention can also be found in combination withtypical compositional formulations of nucleic acids, including in thepresence of carriers, buffers, adjuvants, excipients, diluents, and thelike, as are known to those of ordinary skill in the art.

Unless otherwise indicated, a particular nucleic acid sequence describedherein also implicitly encompasses conservatively modified variantsthereof (e.g., degenerate codon substitutions) and complementarysequences and as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al. (1991) Nucl. Acid Res. 19:5081; Ohtsuka et al.(1985) J. Biol. Chem. 260:2605-2608; Cassol et al. (1992); Rossolini etal. (1994) Mol. Cell. Probes 8:91-98).

Nucleic Acid Hybridization

As noted above, the invention includes nucleic acids that hybridize to atarget nucleic acid of the invention, such as, e.g., a polynucleotideselected from the group consisting of SEQ ID NOS:80-158, 201-204, 223,and 224 or a complementary polynucleotide sequence thereof, whereinhybridization is over substantially the entire length of the targetnucleic acid. The hybridizing nucleic acid may hybridize to a nucleotidesequence of the invention, such as, e.g., that of SEQ ID NO:80, under atleast stringent conditions or under at least high stringency conditions.Moderately stringent, stringent, and highly stringent hybridizationconditions for nucleic acid hybridization experiments are known.Examples of factors that can be combined to achieve such levels ofstringency are briefly discussed herein.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well-characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in P. Tijssen (1993) LABORATORY TECHNIQUES INBIOCHEMISTRY AND MOLECULAR BIOLOGY—HYBRIDIZATION WITH NUCLEIC ACIDPROBES, vol. 24, part I, chapter 2, “Overview of principles ofhybridization and the strategy of nucleic acid probe assays,” (Elsevier,N.Y.) (hereinafter “Tijssen”), as well as in Ausubel, supra, Hames andHiggins (1995) GENE PROBES 1, IRL Press at Oxford University Press,Oxford, England (Hames and Higgins 1) and Hames and Higgins (1995) GENEPROBES 2, IRL Press at Oxford University Press, Oxford, England (Hamesand Higgins 2) provide details on the synthesis, labeling, detection andquantification of DNA and RNA, including oligonucleotides.

An indication that two nucleic acid sequences are substantiallyidentical is that the two molecules hybridize to each other under atleast stringent conditions. The phrase “hybridizing specifically to,”refers to the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetpolynucleotide sequence.

“Stringent hybridization wash conditions” and “stringent hybridizationconditions” in the context of nucleic acid hybridization experiments,such as Southern and northern hybridizations, are sequence dependent,and are different under different environmental parameters. An extensiveguide to hybridization of nucleic acids is found in Tijssen (1993),supra, and in Hames and Higgins 1 and Hames and Higgins 2, supra.

Generally, high stringency conditions are selected such thathybridization occurs at about 5° C. or less than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength andpH. The T_(m) is the temperature (under defined ionic strength and pH)at which 50% of the test sequence hybridizes to a perfectly matchedprobe. In other words, the T_(m) indicates the temperature at which thenucleic acid duplex is 50% denatured under the given conditions and itsrepresents a direct measure of the stability of the nucleic acid hybrid.Thus, the T_(m) corresponds to the temperature corresponding to themidpoint in transition from helix to random coil; it depends on length,nucleotide composition, and ionic strength for long stretches ofnucleotides. Typically, under “stringent conditions,” a probe willhybridize to its target subsequence, but to no other sequences. “Verystringent conditions” are selected to be equal to the T_(m) for aparticular probe.

The T_(m) of a DNA-DNA duplex can be estimated using equation (1): T_(m)(° C.)=81.5° C.+16.6 (log₁₀M)+0.41 (% G+C)−0.72 (% f)−500/n, where M isthe molarity of the monovalent cations (usually Na+), (% G+C) is thepercentage of guanosine (G) and cytosine (C) nucleotides, (% f) is thepercentage of formalize and n is the number of nucleotide bases (i.e.,length) of the hybrid. See Rapley, R. and Walker, J. M. eds., MOLECULARBIOMETHODS HANDBOOK (1998), Humana Press, Inc. (hereinafter Rapley andWalker), Tijssen (1993), supra. The T_(m) of an RNA-DNA duplex can beestimated using equation (2): T_(m) (° C.)=79.8° C.+18.5 (log₁₀M)+0.58(% G+C)-11.8(% G+C)²−0.56 (% f)−820/n, where M is the molarity of themonovalent cations (usually Na+), (% G+C) is the percentage of guanosine(G) and cytosine (C) nucleotides, (% f) is the percentage of formamideand n is the number of nucleotide bases (i.e., length) of the hybrid.Id. Equations 1 and 2 above are typically accurate only for hybridduplexes longer than about 100-200 nucleotides. Id. The T_(m) of nucleicacid sequences shorter than 50 nucleotides can be calculated as follows:T_(m) (° C.)=4(G+C)+2(A+T), where A (adenine), C, T (thymine), and G arethe numbers of the corresponding nucleotides.

In general, non-hybridized nucleic acid material is removed by a seriesof washes, the stringency of which can be adjusted depending upon thedesired results, in conducting hybridization analysis. Low stringencywashing conditions (e.g., using higher salt and lower temperature)increase sensitivity, but can product nonspecific hybridization signalsand high background signals. Higher stringency conditions (e.g., usinglower salt and higher temperature that is closer to the hybridizationtemperature) lower the background signal, typically with only thespecific signal remaining. Addition useful guidance concerning suchhybridization techniques is provided in, e.g., Rapley and Walker, supra(in particular, with respect to such hybridization experiments, part I,chapter 2, “Overview of principles of hybridization and the strategy ofnucleic acid probe assays”), Elsevier, N.Y., as well as in Ausubel,supra, Sambrook, supra, Watson, supra, Hames and Higgins (1995) GENEPROBES 1, IRL Press at Oxford Univ. Press, Oxford, England, and Hamesand Higgins (1995) GENE PROBES 2, IRL Press, Oxford Univ. Press, Oxford,England.

Exemplary stringent (or regular stringency) conditions for analysis ofat least two nucleic acids comprising at least 100 nucleotides includeincubation in a solution or on a filter in a Southern or northern blotcomprises 50% formalin (or formamide) with 1 milligram (mg) of heparinat 42° C., with the hybridization being carried out overnight. A regularstringency wash can be carried out using, e.g., a solution comprising0.2×SSC wash at about 65° C. for about 15 minutes (see Sambrook, supra,for a description of SSC buffer). Often, the regular stringency wash ispreceded by a low stringency wash to remove background probe signal. Alow stringency wash can be carried out in, for example, a solutioncomprising 2×SSC at about 40° C. for about 15 minutes. A highlystringent wash can be carried out using a solution comprising 0.15 MNaCl at about 72° C. for about 15 minutes. An example medium (regular)stringency wash, less stringent than the regular stringency washdescribed above, for a duplex of, e.g., more than 100 nucleotides, canbe carried out in a solution comprising 1×SSC at 45° C. for 15 minutes.An example low stringency wash for a duplex of, e.g., more than 100nucleotides, is carried out in a solution of 4-6×SSC at 40° C. for 15minutes. For short probes (e.g., about 10-50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1.0M Na⁺ ion, typically about 0.01 to 1.0 M Na⁺ ion concentration (or othersalts) at pH 7.0 to 8.3, and the temperature is typically at least about30° C. Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide.

Exemplary moderate stringency conditions include overnight incubation at37° C. in a solution comprising 20% formalin (or formamide), 0.5×SSC, 50mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C., or substantiallysimilar conditions, e.g., the moderately stringent conditions describedin Sambrook, supra, and/or Ausubel, supra.

High stringency conditions are conditions that use, for example, (1) lowionic strength and high temperature for washing, such as 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at50° C., (2) employ a denaturing agent during hybridization, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin (BSA)/0.1% Ficoll/0.1% polyvinylpyrrolidone (PVP)/50 mM sodiumphosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodiumcitrate at 42° C., or (3) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at (i)42° C. in 0.2×SSC, (ii) at 55° C. in 50% formamide and (iii) at 55° C.in 0.1×SSC (preferably in combination with EDTA).

In general, a signal to noise ratio of 2× or 2.5×-5× (or higher) thanthat observed for an unrelated probe in the particular hybridizationassay indicates detection of a specific hybridization. Detection of atleast stringent hybridization between two sequences in the context ofthe present invention indicates relatively strong structural similarityor homology to, e.g., the nucleic acids of the present invention.

As noted, “highly stringent” conditions are selected to be about 5° C.or less lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. Target sequences that areclosely related or identical to the nucleotide sequence of interest(e.g., “probe”) can be identified under highly stringency conditions.Lower stringency conditions are appropriate for sequences that are lesscomplementary. See, e.g., Rapley and Walker; Sambrook, all supra.

Comparative hybridization can be used to identify nucleic acids of theinvention, and this comparative hybridization method is a preferredmethod of distinguishing nucleic acids of the invention. Detection ofhighly stringent hybridization between two nucleotide sequences in thecontext of the present invention indicates relatively strong structuralsimilarity/homology to, e.g., the nucleic acids provided in the sequencelisting herein. Highly stringent hybridization between two nucleotidesequences demonstrates a degree of similarity or homology of structure,nucleotide base composition, arrangement or order that is greater thanthat detected by stringent hybridization conditions. In particular,detection of highly stringent hybridization in the context of thepresent invention indicates strong structural similarity or structuralhomology (e.g., nucleotide structure, base composition, arrangement ororder) to, e.g., the nucleic acids provided in the sequence listingherein. For example, it is desirable to identify test nucleic acids thathybridize to the exemplar nucleic acids herein under stringentconditions.

Thus, one measure of stringent hybridization is the ability to hybridizeto a nucleic acid of the invention (e.g., a nucleic acid comprising apolynucleotide sequence selected from the group consisting of SEQ IDNOS:80-158, 201-204, 223, and 224, or a complementary polynucleotidesequence thereof) under highly stringent conditions (or very stringentconditions, or ultra-high stringency hybridization conditions, orultra-ultra high stringency hybridization conditions). Stringenthybridization (including, e.g., highly stringent, ultra-high stringency,or ultra-ultra high stringency hybridization conditions) and washconditions can easily be determined empirically for any test nucleicacid.

For example, in determining highly stringent hybridization and washconditions, the hybridization and wash conditions are graduallyincreased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration and/or increasing theconcentration of organic solvents, such as formalin, in thehybridization or wash), until a selected set of criteria is met. Forexample, the hybridization and wash conditions are gradually increaseduntil a probe comprising one or more nucleic acid sequences selectedfrom the group consisting of SEQ ID NOS:80-158, 201-204, 223, and 224and complementary polynucleotide sequences thereof, binds to a perfectlymatched complementary target (again, a nucleic acid comprising at leastone nucleic acid sequences selected from the group consisting of SEQ IDNOS:80-158, 201-204, 223, and 224 or complementary polynucleotidesequences thereof), with a signal to noise ratio that is at least 2.5×,and optionally 5× or more as high as that observed for hybridization ofthe probe to an unmatched target. The unmatched target may comprise anucleic acid corresponding to, e.g., a CTLA-4 polypeptide-encodingnucleic acid sequence.

Usually, the hybridization analysis is carried out under hybridizationconditions selected such that a nucleic acid comprising a sequence thatis perfectly complementary to the a disclosed reference (or known)nucleotide sequence (e.g., SEQ ID NO:80) hybridizes with the recombinantantigen-encoding sequence (e.g., a nucleotide sequence variant of thenucleic acid sequence of SEQ ID NO:80) with at least about 5, 7, or 10times higher signal-to-noise ratio than is observed in the hybridizationof the perfectly complementary nucleic acid to a nucleic acid thatcomprises a nucleotide sequence that is at least about 80 or 90%identical to the reference nucleic acid. Such conditions can beconsidered indicative for specific hybridization.

The above-described hybridization conditions can be adjusted, oralternative hybridization conditions selected, to achieve any desiredlevel of stringency in selection of a hybridizing nucleic acid sequence.For example, the above-described highly stringent hybridization and washconditions can be gradually increased (e.g., by increasing temperature,decreasing salt concentration, increasing detergent concentration and/orincreasing the concentration of organic solvents, such as formalin, inthe hybridization or wash), until a selected set of criteria are met.For example, the hybridization and wash conditions can be graduallyincreased until a desired probe, binds to a matched complementarytarget, with a signal-to-noise ratio that is at least about 2.5×, andoptionally at least about 5× (e.g., about 10×, about 20×, about 50×,about 100×, or even about 500×), as high as the signal-to-noise rationobserved from hybridization of the probe to a nucleic acid not of theinvention, such as a nucleic acid encoding WT CTLA-4 ECD polypeptide.

Making and Modifying Nucleic Acids

Nucleic acids of the invention can be obtained and/or generated byapplication of any suitable synthesis, manipulation, and/or isolationtechniques, or combinations thereof. Exemplary procedures are describedinfra. For example, polynucleotides of the invention are typicallyproduced through standard nucleic acid synthesis techniques, such assolid-phase synthesis techniques known in the art. In such techniques,fragments of up to about 100 bases usually are individually synthesized,then joined (e.g., by enzymatic or chemical ligation methods, orpolymerase mediated recombination methods) to form essentially anydesired continuous nucleic acid sequence. The synthesis of the nucleicacids of the invention can be also facilitated (or alternativelyaccomplished), by chemical synthesis using, e.g., the classicalphosphoramidite method, which is described in, e.g., Beaucage et al.(1981) Tetrahedron Letters 22:1859-69, or the method described byMatthes et al. (1984) EMBO J. 3:801-05, e.g., as is typically practicedin automated synthetic methods. The nucleic acid of the invention alsocan be produced by use of an automatic DNA synthesizer. Other techniquesfor synthesizing nucleic acids and related principles are described in,e.g., Itakura et al., Annu. Rev. Biochem. 53:323 (1984), Itakura et al.,Science 198:1056 (1984), and Ike et al., Nucl. Acid Res. 11:477 (1983).

Conveniently, custom made nucleic acids can be ordered from a variety ofcommercial sources, such as The Midland Certified Reagent Company(mcrc@oligos.com), the Great American Gene Company (worldwide websiteaddress genco.com), ExpressGen Inc. (worldwide website addressexpressgen.com), Operon Technologies Inc. (Alameda, Calif.). Similarly,custom peptides and antibodies can be custom ordered from any of avariety of sources, e.g., PeptidoGenic (pkim@ccnet.com), HTIBio-products, Inc. (worldwide website address htibio.com), and BMABiomedicals Ltd. (U.K.), Bio.Synthesis, Inc.

Certain nucleotides of the invention may also be obtained by screeningcDNA libraries using oligonucleotide probes that can hybridize to orPCR-amplify polynucleotides which encode the polypeptides of theinvention. Procedures for screening and isolating cDNA clones and PCRamplification procedures are well known to those of skill in the art;exemplary procedures are described infra (see, e.g., proceduresdescribed in the Examples below). Such techniques are described in,e.g., Berger and Kimmel, “Guide to Molecular Cloning Techniques,” inMethods in Enzymol. Vol. 152, Acad. Press, Inc., San Diego, Calif.(“Berger”); Sambrook, supra; and Ausubel, supra. Some nucleic acids ofthe invention can be obtained by altering a naturally occurringbackbone, e.g., by mutagenesis, in vitro recombination (e.g.,shuffling), or oligonucleotide recombination. In other cases, suchpolynucleotides can be made in silico or through oligonucleotiderecombination methods as described in the references cited herein.

Recombinant DNA techniques useful in modification of nucleic acids arewell known in the art (e.g., restriction endonuclease digestion,ligation, reverse transcription and cDNA production, and PCR). Usefulrecombinant DNA technology techniques and principles related thereto areprovided in, e.g., Mulligan (1993) Science 260:926-932, Friedman (1991)THERAPY FOR GENETIC DISEASES, Oxford University Press, Ibanez et al.(1991) EMBO J. 10:2105-10, Ibanez et al. (1992) Cell 69:329-41 (1992),and U.S. Pat. Nos. 4,440,859, 4,530,901, 4,582,800, 4,677,063,4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and 4,810,648,and are more particularly described in Sambrook et al. (1989) MOLECULARCLONING: A LABORATORY MANUAL, Cold Spring Harbor Press, and the thirdedition thereof (2001), Ausubel et al. (1994-1999), Current Protocols inMolecular Biology, Wiley Interscience Publishers (with Greene PublishingAssociates for some editions), Berger, supra, and Watson, supra.

Modified Coding Sequences

Where appropriate, nucleic acids of the invention can be modified toincrease or enhance expression in a particular host by modification ofthe sequence with respect to codon usage and/or codon context, given theparticular host(s) in which expression of the nucleic acid is desired.Codons that are utilized most often in a particular host are calledoptimal codons, and those not utilized very often are classified as rareor low-usage codons (see, e.g., Zhang, S. P. et al. (1991) Gene105:61-72). Codons can be substituted to reflect the preferred codonusage of the host, a process called “codon optimization” or “controllingfor species codon bias.”

Optimized coding sequence comprising codons preferred by a particularprokaryotic or eukaryotic host can be used to increase the rate oftranslation or to produce recombinant RNA transcripts having desirableproperties, such as a longer half-life, as compared with transcriptsproduced from a non-optimized sequence. Techniques for producingcodon-optimized sequences are known (see, e.g., Murray, E. et al. (1989)Nucl. Acids Res. 17:477-508). Translation stop codons can also bemodified to reflect host preference. For example, preferred stop codonsfor S. cerevisiae and mammals are UAA and UGA respectively. Thepreferred stop codon for monocotyledonous plants is UGA, whereas insectsand E. coli prefer to use UAA as the stop codon (see, e.g., Dalphin, M.E. et al. (1996) Nucl. Acids Res. 24:216-218, for discussion). Thearrangement of codons in context to other codons also can influencebiological properties of a nucleic acid sequences, and modifications ofnucleic acids to provide a codon context arrangement common for aparticular host also is contemplated by the inventors. Thus, a nucleicacid sequence of the invention can comprise a codon optimized nucleotidesequence, i.e., codon frequency optimized and/or codon pair (i.e., codoncontext) optimized for a particular species (e.g., the polypeptide canbe expressed from a polynucleotide sequence optimized for expression inhumans by replacement of “rare” human codons based on codon frequency,or codon context, such as by using techniques such as those described inBuckingham et al. (1994) Biochimie 76(5):351-54 and U.S. Pat. Nos.5,082,767, 5,786,464, and 6,114,148). For example, the inventionprovides a nucleic acid comprising a nucleotide sequence variant of SEQID NO:80, wherein the nucleotide sequence variant differs from thenucleotide sequence of SEQ ID NO:80 by the substitution of “rare” codonsfor a particular host with codons commonly expressed in the host, whichcodons encode the same amino acid residue as the substituted “rare”codons in SEQ ID NO:80.

Vectors, Vector Components, and Expression Systems

The present invention also includes recombinant constructs comprisingone or more of the nucleic acids of the invention as broadly describedabove. Such constructs may comprise a vector, such as a plasmid, acosmid, a phage, a virus, a viral particle, a virus-like particle, abacterial artificial chromosome (BAC), a yeast artificial chromosome(YAC), or the like, or a non-replicating vector, such as a liposome,naked or conjugated DNA, DNA-microparticle, into which at least onenucleic acid sequence of the invention has been inserted, in a forwardor reverse orientation. In a particular aspect of this embodiment, theconstruct further comprises one or more regulatory sequences, including,for example, a promoter, operably linked to a nucleic acid sequence ofthe invention (e.g., nucleic acid encoding an isolated or recombinantmutant CTLA-4 ECD polypeptide or dimeric or monomeric mutant CTLA-4-Ig).Large numbers of suitable vectors and promoters are known to those ofskill in the art and are commercially available. In some instances, avector, such as, e.g., a virus or virus-like particle, may also oralternatively include one or more polypeptides of the invention such as,e.g., incorporated into the coat of the virus or virus-like particle.Vectors can be useful as delivery agents for the delivery oradministration to a subject of exogenous genes or proteins. Vectors ofthe present invention, including those described herein, are useful asdelivery agents for the delivery or administration of nucleic acidsand/or polypeptides of the invention.

General texts that describe molecular biological techniques usefulherein, including the use of vectors, promoters, and many other relevanttopics, include Berger, supra, Sambrook (1989), supra, and Ausubel,supra. Examples of techniques sufficient to direct persons of skillthrough in vitro amplification methods, including the polymerase chainreaction (PCR) the ligase chain reaction (LCR), Q∃-replicaseamplification and other RNA polymerase mediated techniques (e.g.,NASBA), e.g., for the production of the homologous nucleic acids of theinvention are found in Berger, Sambrook, and Ausubel, all supra, as wellas Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols: A Guideto Methods and Applications (Innis et al., eds.) Academic Press Inc. SanDiego, Calif. (1990) (“Innis”); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3:81-94; (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177; Guatelli et al. (1990) Proc.Natl. Acad. Sci. USA 87:1874-1878; Lomeli et al. (1989) J. Clin. Chem.35:1826-1831; Landegren et al. (1988) Science 241:1077-1080; Van Brunt(1990) Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4:560-569;Barringer et al. (1990) Gene 89:117-122, and Sooknanan and Malek (1995)Biotechnology 13:563-564.

PCR generally refers to a procedure wherein minute amounts of a specificpiece of nucleic acid (e.g., RNA or DNA) are amplified by methods wellknown in the art (see, e.g., U.S. Pat. No. 4,683,195 and the otherreferences cited above). Generally, sequence information from the endsof the region of interest or beyond is used for design ofoligonucleotide primers. Such primers will be identical or similar insequence to the opposite strands of the template to be amplified. The 5′terminal nucleotides of the opposite strands may coincide with the endsof the amplified material. PCR may be used to amplify specific RNA orspecific DNA sequences, recombinant DNA or RNA sequences, DNA and RNAsequences from total genomic DNA, and cDNA transcribed from totalcellular RNA, bacteriophage or plasmid sequences, etc. PCR is oneexample, but not the only example, of a nucleic acid polymerase reactionmethod for amplifying a nucleic acid test sample comprising the use ofanother (e.g., known) nucleic acid as a primer. Improved methods ofcloning in vitro amplified nucleic acids are described in Wallace etal., U.S. Pat. No. 5,426,039. Improved methods of amplifying largenucleic acids by PCR are summarized in Cheng et al. (1994) Nature369:684 685 and the references cited therein, in which PCR amplicons ofup to 40 kilobases (kb) are generated. One of skill will appreciate thatessentially any RNA can be converted into a double-stranded DNA suitablefor restriction digestion, PCR expansion and sequencing using reversetranscriptase and a polymerase. See Ausubel, Sambrook, and Berger, allsupra.

The nucleic acids of the present invention can be incorporated into anyone of a variety of vectors, e.g., expression vectors, for expressing apolypeptide, including, e.g., a polypeptide of the invention. Expressionvectors compatible with prokaryotic host cells may be used; suchprokaryotic expression vectors are known in the art and commerciallyavailable. Such vectors include, but are not limited to, e.g.,BLUESCRIPT vector (Stratagene), T7 expression vector (Invitrogen), pETvector (Novagen), and similar prokaryotic vectors.

Expression vectors compatible with eukaryotic host cells mayalternatively be used; such eukaryotic expression vectors are known inthe art and commercially available. Such vectors include, but are notlimited to, e.g., pCMV vectors (e.g., Invitrogen), pIRES vector(Clontech), pSG5 vector (Stratagene), pcDNA3.1 (Invitrogen LifeTechnologies), pcDNA3 (Invitrogen Life Technologies), UbiquitousChromatin Opening Element (UCOE™) expression vector (Millipore), andsimilar eukaryotic expression vectors. The UCOE™ vector is typicallyused for protein production in mammalian cells (e.g., CHO cells).According to Millipore, the UCOE™ expression technology thwartstransgene silencing and provides stable high-level gene expressionwithout respect to the site of chromosomal integration. See Milliporewebsite at worldwide web address millipore.com. An exemplary UCOEexpression vector into which, for example, a nucleic acid of theinvention can be incorporated is the UCOE expression vector namedCET1019AS-puro-SceI, which is available for licensing from Millipore.Information about the UCOE expression vector CET1019AS-puro-SceI can befound in, e.g., John Wynne, “UCOE™ Technology Maximizes ProteinExpression,” BioProcess International 4(7):104-105 (July/August 2006)(RP1725EN00) (available at worldwide web addressmillipore.com/bibliography/tech1/rp1725en00); additional informationabout this vector and licensing of this vector from Millipore can befound the Millipore website, including at, e.g., worldwide web addressesmillipore.com/company/cp3/ucoe_licensing andmillipore.com/techpublications/tech1/ps1013en00. Thus, for example, aDNA sequence encoding a mutant CTLA-4 ECD (e.g., SEQ ID NO:36 or SEQ IDNO:50) fused to a DNA sequence encoding an IgG2 Fc polypeptide (e.g.,SEQ ID NO:184), resulting in the DNA sequence of SEQ ID NO:201 isinserted into a UCOE CET1019AS vector (Millipore) and the resulting DNAplasmid can be used for transfections of host cells.

Expression vectors include chromosomal, nonchromosomal and synthetic DNAsequences, e.g., derivatives of SV40, bacterial vectors (e.g., S.typhimurium, S. typhi, S. flexneri, Listeria monocytogenes, B.anthracis); plasmids; bacterial plasmids; phage DNA; baculovirus; yeastplasmids; vectors derived from combinations of plasmids and phage DNA;viral DNA or RNA vectors, including, e.g., vaccinia virus,adeno-associated virus (AAV), adenovirus, Semliki-Forest virus (e.g.,Notka et al., Biol. Chem. 380:341-52 (1999), pox virus (e.g., MVA),alphavirus (e.g., Venezuelan equine encephalitis virus (VEE), Westernequine encephalitis virus (WEE), Eastern equine encephalitis virus(EEE)), vesicular stomatitis virus (VSV), fowl pox virus, pseudorabies,herpes simplex viruses, retroviruses, and many others. Any vector thattransduces genetic material into a cell, and, if replication is desired,which is replicable and viable in the relevant host can be used. Viraland bacterial vectors serving as delivery vehicles can be attenuated;attenuation should be sufficient to decrease if not eliminate inductionof undesirable disease symptoms. FIG. 1 is a schematic diagram of anexemplary plasmid expression vector pcDNA mutant CTLA-4-Ig which encodesa mutant CTLA-4-Ig of the invention. Additional details regardingsuitable expression vectors are provided below, including in theExamples.

A vector of the invention comprising a nucleic acid sequence of theinvention as described herein, as well as an appropriate promoter orcontrol sequence, can be employed to transform an appropriate host topermit the host to express the protein. Examples of appropriateexpression hosts include: bacterial cells, such as E. coli,Streptomyces, and Salmonella typhimurium; fungal cells, such asSaccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; insectcells such as Drosophila and Spodoptera frugiperda; mammalian cells,such as Chinese Hamster Ovary (CHO) (e.g., CHO-K1), COS (e.g., COS-1,COS-7), baby hamster kidney (BHK), and Human Embryonic Kidney (HEK)(e.g., HEK 293), Bowes melanoma cells, and plant cells. It is understoodthat not all cells or cell lines need to be capable of producing fullyfunctional polypeptides of the invention or fragments thereof. Theinvention is not limited by the host cells employed. Additional detailsregarding suitable host cells are provided below.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the desired polypeptide or fragmentthereof. For example, when large quantities of a particular polypeptideor fragments thereof are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be desirable. Such vectors include, but are not limited to,multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which nucleotide coding sequence of interest(e.g., nucleotide sequence encoding a recombinant mutant CTLA-4-Ig) maybe ligated into the vector in-frame with sequences for theamino-terminal Met and the subsequent 7 residues of beta-galactosidaseso that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster(1989) J. Biol. Chem. 264:5503-5509); pET vectors (Novagen, MadisonWis.); and the like.

Similarly, in the yeast Saccharomyces cerevisiae, a number of vectorscomprising constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used for production of the polypeptidesof the invention. For reviews, see Ausubel, supra, Berger, supra, andGrant et al. (1987) Meth. Enzymol. 153:516-544.

In mammalian host cells, a number of expression systems, such asviral-based systems, may be utilized. In cases where an adenovirus isused as an expression vector, a coding sequence is optionally ligatedinto an adenovirus transcription/translation complex consisting of thelate promoter and tripartite leader sequence. Insertion in anonessential E1 or E3 region of the viral genome results in a viablevirus capable of expressing a polypeptide of interest in infected hostcells (Logan and Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659).In addition, transcription enhancers, such as the Rous sarcoma virus(RSV) enhancer, are used to increase expression in mammalian host cells.

A vector, e.g., expression vector, or polynucleotide of the inventioncan comprise one or more expression control sequences. An expressioncontrol sequence is typically associated with and/or operably linked toa nucleic acid sequence of the invention, such as a nucleic acidencoding a recombinant mutant CTLA-4 ECD polypeptide or recombinantmutant CTLA-4-Ig fusion protein. An expression control sequence istypically a nucleotide sequence that promotes, enhances, or controlsexpression (typically transcription) of another nucleotide sequence.Suitable expression control sequences that may be employed include apromoter, including a constitutive promoter, inducible promoter, and/orrepressible promoter, an enhancer for amplifying expression, aninitiation sequence, a termination translation sequence, a splicingcontrol sequence, and the like.

When a nucleic acid of the invention is included in a vector, thenucleic acid is typically operatively linked to an appropriatetranscription control sequence (promoter) to direct mRNA synthesis.Promoters exert a particularly important impact on the level ofrecombinant polypeptide expression. Any suitable promoter can beutilized. Examples of suitable promoters include the cytomegalovirus(CMV) promoter with or without the first intron (intron A), the HIV longterminal repeat promoter, the phosphoglycerate kinase (PGK) promoter,Rous sarcoma virus (RSV) promoters, such as RSV long terminal repeat(LTR) promoters, SV40 promoters, mouse mammary tumor virus (MMTV)promoters, HSV promoters, such as the Lap2 promoter or the herpesthymidine kinase promoter (as described in, e.g., Wagner et al. (1981)Proc. Natl. Acad. Sci. 78:144-145), promoters derived from SV40 orEpstein Barr virus, adeno-associated viral (AAV) promoters, such as thep5 promoter, metallothionein promoters (e.g., the sheep metallothioneinpromoter or the mouse metallothionein promoter (see, e.g., Palmiter etal. (1983) Science 222:809-814), the human ubiquitin C promoter, E. colipromoters, such as the lac and trp promoters, phage lambda P_(L)promoter, and other promoters known to control expression of genes inprokaryotic or eukaryotic cells (either directly in the cell or inviruses which infect the cell). Promoters that exhibit strongconstitutive baseline expression in mammals, particularly humans, suchas CMV promoters, such as the CMV immediate-early promoter (describedin, e.g., U.S. Pat. Nos. 5,168,062, 5,385,839, 5,688,688, and5,658,759), and promoters having substantial sequence identity with suchCMV promoters, can be employed. Recombinant promoters having enhancedproperties, such as in Int'l Pat. Publ. No. WO 02/00897, may also beused.

A promoter that is operably linked to a nucleic acid of the inventionfor expression of the nucleic acid can have any suitable mechanism ofaction. Thus, the promoter can be, for example, an “inducible” promoter,(e.g., a growth hormone promoter, metallothionein promoter, heat shockprotein promoter, E1B promoter, hypoxia induced promoter, radiationinducible promoter, or adenoviral MLP promoter and tripartite leader),an inducible-repressible promoter, a developmental stage-relatedpromoter (e.g., a globin gene promoter), or a tissue specific promoter(e.g., a smooth muscle cell α-actin promoter, myosin light-chain 1Apromoter, or vascular endothelial cadherin promoter). Suitable induciblepromoters include ecdysone and ecdysone-analog-inducible promoters.Ecdysone-analog-inducible promoters are commercially available, e.g.,through Stratagene (La Jolla, Calif.). If desired, a nucleic acid of theinvention can be induced by using an inducible on- and off-geneexpression system. Examples of such on- and off-gene expression systemsinclude the Tet-On™ Gene Expression System and Tet-Off™ Gene ExpressionSystem, respectively (Clontech, Palo Alto, Calif.; see, e.g., ClontechCatalog 2000, pg. 110-111 for a detailed description of each suchsystem). The inducible promoter can be any promoter that is up- and/ordownregulated in response to an appropriate signal. Additional induciblepromoters include arabinose-inducible promoters, a steroid-induciblepromoters (e.g., a glucocorticoid-inducible promoters), as well as pH,stress, and heat-inducible promoters.

The promoter can be, and often is, a host-native promoter, or a promoterderived from a virus that infects a particular host (e.g., a human betaactin promoter, human EF1α promoter, or a promoter derived from a humanAAV operably linked to the nucleic acid of interest), particularly wherestrict avoidance of gene expression silencing due to host immunologicalreactions to sequences that are not regularly present in the host is ofconcern. A bi-directional promoter system (as described in, e.g., U.S.Pat. No. 5,017,478) linked to multiple nucleotide sequences of interestcan also be utilized.

Other suitable promoters and principles related to the selection, use,and construction of suitable promoters are provided in, e.g., Werner(1999) Mamm Genome 10(2):168-75, Walther et al. (1996) J. Mol. Med.74(7):379-92, Novina (1996) Trends Genet. 12(9):351-55, Hart (1996)Semin. Oncol. 23(1):154-58, Gralla (1996) Curr. Opin. Genet. Dev.6(5):526-30, Fassler et al. (1996) Methods Enzymol 273:3-29, Ayoubi etal. (1996), 10(4) FASEB J 10(4):453-60, Goldsteine et al. (1995)Biotechnol. Annu. Rev. 1:105-28, Azizkhan et al. (1993) Crit. Rev.Eukaryot. Gene Expr. 3(4):229-54, Dynan (1989) Cell 58(1):1-4, Levine(1989) Cell 59(3):405-8, and Berk et al. (1986) Annu. Rev. Genet.20:45-79, as well as U.S. Pat. No. 6,194,191. Other suitable promoterscan be identified by use of the Eukaryotic Promoter Database (release68) (available at the worldwide website address epd.isb-sib.ch/) andother similar databases, such as the Transcription Regulatory RegionsDatabase (TRRD) (version 4.1) (available at the worldwide websiteaddress bionet.nsc.ru/trrd/) and the transcription factor database(TRANSFAC) (available at the worldwide website addresstransfac.gbf.de/TRANSFAC/index.html).

As an alternative to a promoter, particularly in RNA vectors andconstructs, a vector or nucleic acid of the invention can comprise oneor more internal ribosome entry sites (IRESs), IRES-encoding sequences,or RNA sequence enhancers (Kozak consensus sequence analogs), such asthe tobacco mosaic virus omega prime sequence.

A vector or polynucleotide of the invention can include an upstreamactivator sequence (UAS), such as a Gal4 activator sequence (see, e.g.,U.S. Pat. No. 6,133,028) or other suitable upstream regulatory sequence(see, e.g., U.S. Pat. No. 6,204,060).

A vector or polynucleotide of the invention can include a Kozakconsensus sequence that is functional in a mammalian cell. The Kozaksequence can be a naturally occurring or modified sequence, such as themodified Kozak consensus sequences described in U.S. Pat. No. 6,107,477.

Specific initiation signals can aid in efficient translation of a codingsequence of the invention, such as a mutant CTLA-4 ECDpolypeptide-encoding nucleotide sequence. Such signals can be includedin a vector of the invention. These signals can include, e.g., the ATGinitiation codon and adjacent sequences. In cases where a codingsequence, its initiation codon, and upstream sequences are inserted intoan appropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only a coding sequence(e.g., a mature protein coding sequence), or a portion thereof isinserted, exogenous nucleic acid transcriptional control signalsincluding the ATG initiation codon must be provided. Furthermore, theinitiation codon must be in the correct reading frame to ensuretranscription of the entire insert. Exogenous transcriptional elementsand initiation codons can be of various origins—both natural andsynthetic. The efficiency of expression can be enhanced by the inclusionof enhancers appropriate to the cell system in use (see, e.g., Scharf etal., Results Probl. Cell. Differ. 20:125-62 (1994); and Bittner et al.,Meth. Enzymol. 153:516-544 (1987)). Suitable enhancers include the Roussarcoma virus (RSV) enhancer and the RTE enhancers described in U.S.Pat. No. 6,225,082.

The skilled artisan will recognize that the introduction of a startcodon (ATG) to the 5′ end of a particular nucleotide sequence ofinterest usually results in the addition of an N-terminal methionine tothe encoded amino acid sequence when the sequence is expressed in amammalian cell (other modifications may occur in bacterial and/or othereukaryotic cells, such as introduction of an formyl-methionine residueat a start codon). For expression of a nucleic acid of the invention ineukaryotic cells, a start codon and a nucleotide sequence encoding asignal peptide are typically be included at the 5′ end of a nucleic acidsequence of the invention (e.g., SEQ ID NO:80), and a termination codonis typically included at the C terminus of the nucleic acid (e.g., SEQID NO:80). An exemplary signal peptide sequence is the hCTLA-4 signalpeptide sequence (SEQ ID NO:182); the nucleic acid sequence encoding thetissue plasminogen activator signal peptide is shown in SEQ ID NO:181.Another exemplary signal peptide sequence is the hCTLA-4 signal peptidesequence (SEQ ID NO:216), which is encoded by the nucleic acid sequenceshown in SEQ ID NO:215.

Termination sequences are discussed in detail below.

Such elements can be included in the vector construct of choice. Uponexpression, the polypeptide variant encoded by the nucleic acid (e.g.,SEQ ID NO:80) will initially include an N-terminal methionine residueand the signal peptide sequence. However, the N-terminal methionine andsignal peptide sequence will be cleaved upon secretion, therebygenerating the encoded polypeptide (e.g., SEQ ID NO:1).

The expression level of a nucleic acid of the invention (or acorresponding polypeptide of the invention can be assessed by anysuitable technique. Examples include Northern Blot analysis (discussedin, e.g., McMaster et al., Proc. Natl. Acad. Sci. USA 74(11):4835-38(1977) and Sambrook, infra), reverse transcriptase-polymerase chainreaction (RT-PCR) (as described in, e.g., U.S. Pat. No. 5,601,820 andZaheer et al., Neurochem. Res. 20:1457-63 (1995)), and in situhybridization techniques (as described in, e.g., U.S. Pat. Nos.5,750,340 and 5,506,098). Quantification of proteins also can beaccomplished by the Lowry assay and other protein quantification assays(see, e.g., Bradford, Anal. Biochem. 72:248-254 (1976); Lowry et al., J.Biol. Chem. 193:265 (1951)). Western blot analysis of recombinantpolypeptides of the invention obtained from the lysate of cellstransfected with polynucleotides encoding such recombinant polypeptidesis another suitable technique for assessing levels of recombinantpolypeptide expression.

A vector, e.g., expression vector, or polynucleotide of the inventioncan comprise a ribosome-binding site for translation initiation and atranscription-terminating region. A suitable transcription-terminatingregion is, for example, a polyadenylation sequence that facilitatescleavage and polyadenylation of an RNA transcript produced from a DNAsequence. Any suitable polyadenylation sequence can be used, including asynthetic optimized sequence, as well as the polyadenylation sequence ofBGH (Bovine Growth Hormone), human growth hormone gene, polyoma virus,TK (Thymidine Kinase), EBV (Epstein Barr Virus), rabbit beta globin, andthe papillomaviruses, including human papillomaviruses and BPV (BovinePapilloma Virus). Suitable polyadenylation (polyA) sequences alsoinclude the SV40 (human Sarcoma Virus-40) polyadenylation sequence andthe BGH polyA sequence. Such polyA sequences are described in, e.g.,Goodwin et al. (1998) Nucleic Acids Res. 26(12):2891-8, Schek et al.(1992) Mol. Cell. Biol. 12(12):5386-93, and van den Hoff et al. (1993)Nucleic Acids Res. 21(21):4987-8. Additional principles related toselection of appropriate polyadenylation sequences are described in,e.g., Levitt et al. (1989) Genes Dev. 1989 3(7):1019-1025, Jacob et al.(1990) Crit. Rev. Eukaryot. Gene Expr. 1(1):49-59, Chen et al. (1995)Nucleic Acids Res. 23(14):2614-2620, Moreira et al. (1995) EMBO J.14(15):3809-3819, Carswell et al. (1989) Mol. Cell. Biol.9(10):4248-4258.

A vector or polynucleotide of the invention can further comprisesite-specific recombination sites, which can be used to modulatetranscription of a nucleotide sequence of interest, as described in,e.g., U.S. Pat. Nos. 4,959,317, 5,801,030 and 6,063,627, European PatentApplication No. 0 987 326 and Int'l Patent App. Publ. No. WO 97/09439.

A vector or polynucleotide of the invention can also comprise a nucleicacid encoding a secretion/localization sequence, to target polypeptideexpression to a desired cellular compartment, membrane, or organelle, orto direct polypeptide secretion to the periplasmic space or into thecell culture media. Such sequences are known in the art, and includesecretion leader peptides or signal peptides, organelle targetingsequences (e.g., nuclear localization sequences, ER retention signals,mitochondrial transit sequences, chloroplast transit sequences),membrane localization/anchor sequences (e.g., stop transfer sequences,GPI anchor sequences), and the like. Polynucleotides of the inventioncan be fused, for example, in-frame to such a nucleic acid encoding asecretion and/or localization sequence. Polypeptides expressed by suchpolynucleotides of the invention may include the amino acid sequencecorresponding to the secretion and/or localization sequence(s).

In addition, a vector or polynucleotide of the invention can compriseone or more selectable marker nucleotide sequences or genes to provide aphenotypic trait for selection of transformed host cells, such asdihydrofolate reductase resistance, neomycin resistance, G418resistance, puromycin resistance, and/or blasticidin resistance foreukaryotic cell culture, or such as tetracycline or ampicillinresistance in E. coli.

A vector or polynucleotide of the invention can also comprise an originof replication useful for propagation in a microorganism. The bacterialorigin of replication (Ori) utilized is preferably one that does notadversely affect gene expression in mammalian cells. Examples of usefulorigin of replication sequences include the f1 phage ori, RK2 oriV, pUCori, and the pSC101 ori. Origin of replication sequences include theColEI ori and the p15 (available from plasmid pACYC177, New EnglandBiolab, Inc.), alternatively another low copy ori sequence (similar top15) can be desirable in some contexts. The nucleic acid in this respectdesirably acts as a shuttle vector, able to replicate and/or beexpressed in both eukaryotic and prokaryotic hosts (e.g., a vectorcomprising an origin of replication sequences recognized in botheukaryotes and prokaryotes).

The invention includes a naked DNA or RNA vector, including, forexample, a linear expression element (as described in, e.g., Sykes andJohnston (1997) Nat Biotech 17:355-59), a compacted nucleic acid vector(as described in, e.g., U.S. Pat. No. 6,077,835 and/or Int'l Pat. App.Publ. No. WO 00/70087), a plasmid vector such as pcDNA3.1, pBR322, pUC19/18, or pUC 118/119, a “midge” minimal-sized nucleic acid vector (asdescribed in, e.g., Schakowski et al. (2001) Mol. Ther. 3:793-800) or asa precipitated nucleic acid vector construct, such as a CaPO₄precipitated construct (as described in, e.g., Int'l Patent Appn WO00/46147, Benvenisty and Reshef (1986) Proc. Natl. Acad. Sci. USA83:9551-55, Wigler et al. (1978), Cell 14:725, and Coraro and Pearson(1981) Somatic Cell Genetics 7:603), comprising a nucleic acid of theinvention. For example, the invention provides a naked DNA plasmidcomprising SEQ ID NO:80 operably linked to a CMV promoter or CMVpromoter variant and a suitable polyadenylation sequence. Nakednucleotide vectors and the usage thereof are known in the art (see,e.g., U.S. Pat. Nos. 5,589,466 and 5,973,972).

A vector of the invention typically is an expression vector that issuitable for expression in a bacterial system, eukaryotic system,mammalian system, or other system (as opposed to a vector designed forreplicating the nucleic acid sequence without expression, which can bereferred to as a cloning vector). For example, in one aspect, theinvention provides a bacterial expression vector comprising a nucleicacid sequence of the invention (e.g., nucleic acid sequence encoding arecombinant mutant CTLA-4-Ig). Suitable vectors include, for example,vectors which direct high level expression of fusion proteins that arereadily purified (e.g., multifunctional E. coli cloning and expressionvectors such as BLUESCRIPT (Stratagene), pIN vectors (Van Heeke &Schuster, J. Biol. Chem. 264:5503-5509 (1989); pET vectors (Novagen,Madison Wis.); and the like). While such bacterial expression vectorscan be useful in expressing particular polypeptides of the invention,glycoproteins of the invention are preferably expressed in eukaryoticcells and as such the invention also provides eukaryotic expressionvectors.

The expression vector can be a vector suitable for expression of thenucleic acid of the invention in a yeast cell. Any vector suitable forexpression in a yeast system can be employed. Suitable vectors for usein, e.g., Saccharomyces cerevisiae include, e.g., vectors comprisingconstitutive or inducible promoters such as alpha factor, alcoholoxidase and PGH (reviewed in Ausubel, supra, Berger, supra, and Grant etal., Meth. Enzymol. 153:516-544 (1987)). Usually, the expression vectorwill be a vector suitable for expression of a nucleic acid of theinvention in an animal cell, such as an insect cell (e.g., a SF-9 cell)or a mammalian cell (e.g., a CHO cell, 293 cell, HeLa cell, humanfibroblast cell, or similar well-characterized cell). Suitable mammalianexpression vectors are known in the art (see, e.g., Kaufman, Mol.Biotechnol. 16(2):151-160 (2000), Van Craenenbroeck, Eur. J. Biochem.267(18):5665-5678 (2000), Makrides, Protein Expr. Purif. 17(2):183-202(1999), and Yarranton, Curr. Opin. Biotechnol. 3(5):506-511 (1992)).Suitable insect cell plasmid expression vectors also are known (Braun,Biotechniques 26(6):1038-1040:1042 (1999)).

An expression vector typically can be propagated in a host cell, whichmay be a eukaryotic cell (such as a mammalian cell, yeast cell, or plantcell) or a prokaryotic cell, such as a bacterial cell. Introduction of anucleic acid vector or expression vector into the host cell (e.g.,transfection) can be effected by calcium phosphate transfection (see,e.g., calcium phosphate co-precipitation method of Graham et al.,Virology 52:456-457 (1973)), DEAE-Dextran mediated transfection,electroporation, gene or vaccine gun, injection, lipofection andbiolistics or other common techniques (see, e.g., Kriegler, GENETRANSFER AND EXPRESSION: A LABORATORY MANUAL, Stockton Press (1990); seeDavis, L., Dibner, M., and Battey, I., BASIC METHODS IN MOLECULARBIOLOGY (1986) for a description of in vivo, ex vivo, and in vitromethods). Cells comprising these and other vectors of the invention forman important part of the invention.

In one aspect, the invention provides an expression vector comprising:(i) a first polynucleotide sequence that encodes a first polypeptidecomprising a polypeptide sequence having at least 95%, 96% 97%, 98%,99%, or 100% sequence identity to at least one polypeptide sequenceselected from the group consisting of SEQ ID NOS:1-73, wherein saidfirst polypeptide binds human CD86 and/or human CD80 and/or anextracellular domain of either or both, and/or suppresses an immuneresponse, and (ii) a second polynucleotide sequence that encodes asecond polypeptide comprising a hinge region, a CH2 domain, and a CH3domain of an immunoglobulin (Ig) polypeptide. The Ig polypeptide isoptionally a human Ig Fc polypeptide (e.g., IgG1, IgG2, IgG4, etc.) or amutant Ig Fc polypeptide. (e.g., an Ig Fc polypeptide in which one ormore cysteine residues have been substituted with another amino acid(e.g., a serine residue), thereby eliminating one or more disulfidebonds formed between two Ig chains, or in which one or more prolineresidues is substituted with another amino acid (e.g., proline) toreduce effector function (reduced Fc receptor binding). In anotheraspect, the invention provides an expression vector comprising anucleotide sequence encoding a fusion protein having at least 95%, 96%,97%, 98%, 99%, or 100% sequence identity to at least one polypeptidesequence selected from the group consisting of SEQ ID NOS: 74-79,197-200, 205-214, and 219-222.

Additional nucleic acids provided by the invention include cosmids. Anysuitable cosmid vector can be used to replicate, transfer, and expressthe nucleic acid sequence of the invention. Typically, a cosmidcomprises a bacterial oriV, an antibiotic selection marker, a cloningsite, and either one or two cos sites derived from bacteriophage lambda.The cosmid can be a shuttle cosmid or mammalian cosmid, comprising aSV40 oriV and, desirably, suitable mammalian selection marker(s). Cosmidvectors are further described in, e.g., Hohn et al. (1988) Biotechnology10:113-27.

Nucleic acids of the invention can be included in and/or administered toa host or host cell in the form of a suitable delivery vehicle (i.e., avector). The vector can be any suitable vector, including chromosomal,non-chromosomal, and synthetic nucleic acid vectors, or other vectorsdescribed above, and may include any combination of the above-describedexpression elements and/or other transfection-facilitating and/orexpression-promoting sequence elements. Examples of such vectors includeviruses, bacterial plasmids, phages, cosmids, phagemids, derivatives ofSV40, baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors,polylysine, and bacterial cells.

Delivery of a recombinant DNA sequence of the invention can beaccomplished with a naked DNA plasmid or plasmid associated with one ormore transfection-enhancing agents, as discussed further herein. Theplasmid DNA vector can have any suitable combination of features.Plasmid DNA vectors may comprise a strong promoter/enhancer region(e.g., human CMV, RSV, SV40, SL3-3, MMTV, or HIV LTR promoter), aneffective poly(A) termination sequence, an origin of replication forplasmid product in E. coli, an antibiotic resistance gene as selectablemarker, and a convenient cloning site (e.g., a polylinker). A particularplasmid vector for delivery of the nucleic acid of the invention in thisrespect is shown in FIG. 1; the construction and features of this vectorare described in the Examples below.

In another aspect, the invention provides a non-nucleic acid vectorcomprising at least one nucleic acid or polypeptide of the invention.Such a non-nucleic acid vector includes, e.g., but is not limited to, arecombinant virus, a viral nucleic acid-protein conjugate (which, withrecombinant viral particles, may sometimes be referred to as a viralvector), or a cell, such as recombinant (and usually attenuated)Salmonella, Shigella, Listeria, and Bacillus Calmette-Guérin (BCG)bacterial cells. Thus, for example, the invention provides a viralvector, insect vector, bacterial vector, or plant vector comprising anucleic acid of the sequence of the invention. Any suitable viral,insect, plant, or bacterial vector can be used in this respect and anumber are known in the art. A viral vector can comprise any number ofviral polynucleotides, alone (a viral nucleic acid vector) or morecommonly in combination with one or more (typically two, three, or more)viral proteins, which facilitate delivery, replication, and/orexpression of the nucleic acid of the invention in a desired host cell.

In one aspect, intracellular bacteria (e.g., Listeria monocytogenes) canbe used to deliver a nucleic acid of the invention. An exemplarybacterial vector for plasmid DNA delivery of one or more nucleic acidsof the invention is Listeria monocytogenes (Lieberman et al., Vaccine20:2007-2010 (2002)).

The invention includes recombinant or isolated viral vectors that havebeen modified to comprise one or more nucleic acids or polypeptides ofthe invention. A viral vector may include a polynucleotide comprisingall or part of a viral genome, a viral protein/nucleic acid conjugate, avirus-like particle (VLP), a vector similar to those described in U.S.Pat. No. 5,849,586 and Int'l Patent App. Publ. No. WO 97/04748, or anintact virus particle comprising one or more viral nucleic acids, andthe viral vector is typically engineered to include at least one nucleicacid and/or polypeptide of the invention. A viral vector (i.e., arecombinant virus) can comprise a wild-type viral particle or a modifiedviral particle, particular examples of which are discussed below.Numerous viruses are typically used as vectors for the delivery ofexogenous nucleic acids, including at least one nucleic acid of theinvention, such as a nucleic acid encoding a mutant CTLA-4 ECD or mutantCTLA-4-Ig described herein. Such vectors include recombinantly modifiedenveloped or non-enveloped DNA and RNA viruses, typically selected frombaculoviridiae, parvoviridiae, picomoviridiae, herpesveridiae,poxyiridae, adenoviridiae, or picornaviridiae. Viral vectors may bewild-type or may be modified by recombinant nucleic acid techniques tobe replication deficient, replication competent, or conditionallyreplicating.

The viral vector can be a vector that requires the presence of anothervector or wild-type virus for replication and/or expression (i.e., ahelper-dependent virus), such as an adenoviral vector amplicon.Typically, such viral vectors comprise a wild-type viral particle, or aviral particle modified in its protein and/or nucleic acid content toincrease transgene capacity or aid in transfection and/or expression ofthe nucleic acid (examples of such vectors include the herpes virus/AAVamplicons). The viral genome may be modified to include induciblepromoters that achieve replication or expression only under certainconditions.

The viral vector can be derived from or comprise a virus that normallyinfects animals, preferably vertebrates, such as mammals, including,e.g., humans. Suitable viral vector particles in this respect, include,for example, adenoviral vector particles (including any virus of orderived from a virus of the adenoviridae), adeno-associated viral vectorparticles (AAV vector particles) or other parvoviruses and parvoviralvector particles, papillomaviral vector particles, Semliki-Forest viralvector, flaviviral vectors, picornaviral vectors, alphaviral vectors,herpes viral vectors, pox virus vectors, retroviral vectors, includinglentiviral vectors. Examples of such viruses and viral vectors areprovided in, e.g., Fields Virology, supra, Fields et al., eds.,VIROLOGY, Raven Press, Ltd., New York (3rd ed., 1996 and 4th ed., 2001),ENCYCLOPEDIA OF VIROLOGY, R. G. Webster et al., eds., Academic Press(2nd ed., 1999), FUNDAMENTAL VIROLOGY, Fields et al., eds.,Lippincott-Raven (3rd ed., 1995), Levine, “Viruses,” Scientific AmericanLibrary No. 37 (1992), MEDICAL VIROLOGY, D. O. White et al., eds.,Academic Press (2nd ed. 1994), and INTRODUCTION TO MODERN VIROLOGY,Dimock, N. J. et al., eds., Blackwell Scientific Publications, Ltd.(1994).

Viral vectors that can be employed with nucleic acids of the inventionand the methods described herein include adeno-associated virus vectors,which are reviewed in, e.g., Carter (1992) Curr. Opinion Biotech.3:533-539 (1992) and Muzcyzka (1992) Curr. Top. Microbiol. Immunol.158:97-129 (1992). Additional types and aspects of AAV vectors aredescribed in, e.g., Buschacher et al., Blood 5(8):2499-504, Carter,Contrib. Microbiol. 4:85-86 (2000), Smith-Arica, Curr. Cardiol. Rep.3(1):41-49 (2001), Taj, J. Biomed. Sci. 7(4):279-91 (2000), Vigna etal., J. Gene Med. 2(5):308-16 (2000), Klimatcheva et al., Front. Biosci.4:D481-96 (1999), Lever et al., Biochem. Soc. Trans. 27(6):841-47(1999), Snyder, J. Gene Med. 1(3):166-75 (1999), Gerich et al., KneeSurg. Sports Traumatol. Arthrosc. 5(2):118-23 (1998), and During, Adv.Drug Deliv. Review 27(1):83-94 (1997), and U.S. Pat. Nos. 4,797,368,5,139,941, 5,173,414, 5,614,404, 5,658,785, 5,858,775, and 5,994,136, aswell as other references discussed elsewhere herein). Adeno-associatedviral vectors can be constructed and/or purified using the methods setforth, for example, in U.S. Pat. No. 4,797,368 and Laughlin et al., Gene23:65-73 (1983).

Alphavirus vectors can be gene delivery vectors in other contexts.Alphavirus vectors are known in the art and described in, e.g., Carter(1992) Curr Opinion Biotech 3:533-539, Schlesinger Expert Opin. Biol.Ther. (2001) 1(2):177-91, Polo et al., Dev. Biol. (Basel). 2000;104:181-5, Wahlfors et al., Gene Ther. (2000) 7(6):472-80, Int'l Pat.App. Publ. Nos. WO 01/81609, WO 00/39318, WO 01/81553, WO 95/07994, WO92/10578.

Another advantageous group of viral vectors are the herpes viralvectors. Examples are described in, e.g., Lachmann et al., Curr. Opin.Mol. Ther. (1999) 1(5):622-32, Fraefel et al., Adv. Virus Res. (2000)55:425-51, Huard et al., Neuromuscul. Disord. (1997) 7(5):299-313,Frenkel et al., Gene Ther. (1994) Suppl 1:S40-6, U.S. Pat. Nos.6,261,552 and 5,599,691.

Retroviral vectors, including lentiviral vectors, also can beadvantageous gene delivery vehicles in particular contexts. There arenumerous retroviral vectors known in the art. Examples of retroviralvectors are described in, e.g., Miller, Curr Top Microbiol. Immunol.(1992) 158:1-24, Weber et al., Curr. Opin. Mol. Ther. (2001)3(5):439-53, Hu et al., Pharmacol. Rev. (2000) 52(4):493-511, Kim etal., Adv. Virus Res. (2000) 55:545-63, Palu et al., Rev. Med. Virol.(2000) 10(3):185-202, Takeuchi et al., Adv. Exp. Med. Biol. (2000)465:23-35, U.S. Pat. Nos. 6,326,195, 5,888,502, 5,580,766, and5,672,510.

Baculovirus vectors are another advantageous group of viral vectors,particularly for the production of polypeptides of the invention. Theproduction and use of baculovirus vectors is known (see, e.g., Kost,Curr. Opin. Biotechnol. 10(5):428-433 (1999); Jones, Curr. Opin.Biotechnol. 7(5):512-516 (1996)). Where the vector is used fortherapeutic uses, the vector will be selected such that it is able toadequately infect (or in the case of nucleic acid vectors transfect ortransform) target cells in which the desired therapeutic effect isdesired.

Adenoviral vectors also can be suitable viral vectors for gene transfer.Adenoviral vectors are well known in the art and described in, e.g.,Graham et al. (1995) Mol. Biotechnol. 33(3):207-220, Stephenson (1998)Clin. Diagn. Virol. 10(2-3):187-94, Jacobs (1993) Clin Sci (Lond).85(2):117-22, U.S. Pat. Nos. 5,922,576, 5,965,358 and 6,168,941 andInternational Patent Applications WO 98/22588, WO 98/56937, WO 99/15686,WO 99/54441, and WO 00/32754. Adenoviral vectors, herpes viral vectors,and Sindbis viral vectors, useful in the practice of the invention andsuitable for organismal in vivo transduction and expression of nucleicacids of the invention, are generally described in, e.g., Jolly (1994)Cancer Gene Therapy 1:51-64, Latchman (1994) Molec. Biotechnol.2:179-195, and Johanning et al. (1995) Nucl. Acids Res. 23:1495-1501.

The virus vector may be replication-deficient in a host cell.Adeno-associated virus (AAV) vectors, which are naturallyreplication-deficient in the absence of complementing adenoviruses or atleast adenovirus gene products (provided by, e.g., a helper virus,plasmid, or complementation cell), are included. By“replication-deficient” is meant that the viral vector comprises agenome that lacks at least one replication-essential gene function. Adeficiency in a gene, gene function, or gene or genomic region, as usedherein, is defined as a deletion of sufficient genetic material of theviral genome to impair or obliterate the function of the gene whosenucleic acid sequence was deleted in whole or in part.Replication-essential gene functions are those gene functions that arerequired for replication (i.e., propagation) of a replication-deficientviral vector. The essential gene functions of the viral vector particlevary with the type of viral vector particle at issue. Examples ofreplication-deficient viral vector particles are described in, e.g.,Marconi et al., Proc. Natl. Acad. Sci. USA 93(21):11319-20 (1996),Johnson and Friedmann, Methods Cell Biol. 43 (pt. A):211-30 (1994),Timiryasova et al., J. Gene Med. 3(5):468-77 (2001), Burton et al., StemCells 19(5):358-77 (2001), Kim et al., Virology 282(1):154-67 (2001),Jones et al., Virology 278(1):137-50 (2000), Gill et al., J. Med. Virol.62(2):127-39 (2000). Other replication-deficient vectors are based onsimple MLV vectors (Miller et al. (1990) Mol. Cell. Biol. 10:4239;Kolberg (1992) J. NIH Res. 4:43, and Cornetta et al. (1991) Hum. Gene.Ther. 2:215). Canary pox vectors are advantageous in infecting humancells but being naturally incapable of replication therein (i.e.,without genetic modification).

The basic construction of recombinant viral vectors is well understoodin the art and involves using standard molecular biological techniquessuch as those described in, e.g., Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL (Cold Spring Harbor Press 1989) and the third editionthereof (2001), Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY(Wiley Interscience Publishers 1995), and Watson, supra, and several ofthe other references mentioned herein. For example, adenoviral vectorscan be constructed and/or purified using the methods set forth, forexample, in Graham et al., Mol. Biotechnol. 33(3):207-220 (1995), U.S.Pat. No. 5,965,358, Donthine et al., Gene Ther. 7(20):1707-14 (2000),and other references described herein. Adeno-associated viral vectorscan be constructed and/or purified using the methods set forth, forexample, in U.S. Pat. No. 4,797,368 and Laughlin et al., Gene 23:65-73(1983). Similar techniques are known in the art with respect to otherviral vectors, particularly with respect to herpes viral vectors (seee.g., Lachman et al., Curr. Opin. Mol. Ther. 1(5):622-32 (1999)),lentiviral vectors, and other retroviral vectors. In general, the viralvector comprises an insertion of the nucleic acid (for example, awild-type adenoviral vector can comprise an insertion of up to 3 KBwithout deletion), or, more typically, comprises one or more deletionsof the virus genome to accommodate insertion of the nucleic acid andadditional nucleic acids, as desired, and to prevent replication in hostcells.

Non-viral vectors, such as, e.g., DNA plasmids, naked nucleic acids, andnucleic acid complexed with a delivery vehicle such as a liposome, alsocan be associated with molecules that target the vector to a particularregion in the host (e.g., a particular organ, tissue, and/or cell type).For example, a nucleotide can be conjugated to a targeting protein, suchas a viral protein that binds a receptor or a protein that binds areceptor of a particular target (e.g., by modification of the techniquesin Wu et al., J. Biol. Chem. 263(29):14621-24 (1988)). Targeted cationiclipid compositions are known (see, e.g., U.S. Pat. No. 6,120,799). Othertechniques for targeting genetic constructs are provided in Int'l Pat.App. Publ. No. WO 99/41402.

Expression Hosts

The present invention also provides engineered host cells transduced,transfected or transformed with a vector of the invention (e.g., acloning vector or expression vector) or a nucleic acid of the invention.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants, or amplifying the nucleic acid of interest. The cultureconditions, such as temperature, pH, and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothose skilled in the art and in the references cited herein, including,e.g., Freshney (1994) Culture of Animal Cells, a Manual of BasicTechnique, 3rd ed., Wiley-Liss, New York and the references citedtherein. Polypeptides of the invention encoded by such vectors ornucleic acids of the invention are expressed in such host cells and canbe isolated by standard techniques. For example, polypeptides releasedinto the cell culture can be isolated from the culture byultracentrifugation or similar techniques.

The polypeptides of the invention can be produced in a variety ofexpression hosts, including, but not limited to, animal cells, such asmammalian cells (e.g., CHO cells), including human and non-human primatecells, and in non-animal cells, such as plants, yeast, fungi, bacteria,and the like. Examples of suitable expression hosts include bacterialcells, such as E. coli, Streptomyces, and Salmonella typhimurium; fungalcells, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurosporacrassa; insect cells such as Drosophila and Spodoptera frugiperda;mammalian cells, such as CHO (e.g., CHO-K1), COS (e.g., COS-1, COS-7),BHK, and HEK (e.g., HEK 293) cells, Bowes melanoma cells, and plantcells. As noted above, the invention is not limited by the host cellsemployed. In addition to Sambrook, Berger and Ausubel, all supra,details regarding cell culture are found in, e.g., Payne et al. (1992)Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc.New York, N.Y.; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissueand Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg NY); Atlas & Parks (eds.) TheHandbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.Such host cells can be adapted to growth in serum-free, protein-freemedium, animal component-free medium, such as, e.g., a chemicallydefined (CD) medium (such as, e.g., CD OptiCHO™ (Invitrogen, #12681)using procedures known in the art.

The invention provides a cell(s) comprising any one or more of thenucleic acids, vectors, or other constructs of the invention (e.g., aconstruct expressing a mutant CTLA-4 ECD or mutant CTLA-4-Ig) describedherein or any combination thereof. Also included is a cell comprisingone or more of any of the polypeptides, antibodies, or fusion proteins,or other constructs of the invention described herein, or anycombination of one or more of these. A cell of the invention istypically an isolated or recombinant cell and may comprise a host cell.Such a cell, e.g., recombinant cell, may be modified by transformation,transfection, and/or infection with at least one nucleic acid, vector,or other construct of the invention. Such a cell can be a eukaryoticcell (e.g., mammalian, yeast, or plant cell) or a prokaryotic cell(e.g., bacterial cell) and can be transformed with any such construct ofthe invention using a variety of known methods, including, e.g., calciumphosphate transfection (see, e.g., calcium phosphate co-precipitationmethod), DEAE-Dextran mediated transfection, electroporation (Irving etal., Cell 64:891-901 (1991)), gene or vaccine gun, injection,lipofection and biolistics or other common techniques as noted above.See also Inovio Biomedical Corp. electroporation methods and technologyat the worldwide website address inovio.com.

A host cell strain is optionally chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the protein include, butare not limited to, acetylation, carboxylation, glycosylation,phosphorylation, lipidation and acylation. Different host cells such asE. coli, Bacillus sp., yeast, or mammalian cells, such as CHO, HeLa,BHK, MDCK, HEK 293, WI38, etc. have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and maybe chosen to ensure the correct modification and processing of theintroduced foreign protein.

A nucleic acid of the invention can be inserted into an appropriate hostcell (in culture or in a host organism) to permit the host to express aprotein of interest (e.g., mutant CTLA-4 ECD or mutant CTLA-4-Ig). Anysuitable host cell can be transformed/transduced by the nucleic acids ofthe invention. Examples of appropriate expression hosts include:bacterial cells, such as E. coli, Streptomyces, Bacillus sp., andSalmonella typhimurium; fungal cells, such as Saccharomyces cerevisiae,Pichia pastoris, and Neurospora crassa; insect cells such as Drosophilaand Spodoptera frugiperda; mammalian cells such as Vero cells, HeLacells, CHO cells (e.g., CHO-K1), COS cells, WI38 cells, NIH-3T3 cells(and other fibroblast cells, such as MRC-5 cells), MDCK cells, KB cells,SW-13 cells, MCF7 cells, BHK cells, HEK-293 cells, Bowes melanoma cells,and plant cells, etc.

The present invention also provides host cells that are transduced,transformed or transfected with at least one nucleic acid or vector ofthe invention. As discussed above, a vector of the invention typicallycomprises a nucleic acid of the invention. Host cells are geneticallyengineered (e.g., transduced, transformed, infected, or transfected)with the vectors of the invention, which may be, e.g., a cloning vectoror an expression vector. The vector may be in the form of a plasmid, aviral particle, a phage, attenuated bacteria, or any other suitable typeof vector. Host cells suitable for transduction and/or infection withviral vectors of the invention for production of the recombinantpolypeptides of the invention and/or for replication of the viral vectorof the invention include the above-described cells. Examples of cellsthat have been demonstrated as suitable for packaging of viral vectorparticles are described in, e.g., Polo et al., Proc. Natl. Acad. Sci.96(8):4598-603 (1999), Farson et al., J. Gene Med. 1(3):195-209 (1999),Sheridan et al., Mol. Ther. 2(3):262-75 (2000), Chen et al., Gene Ther.8(9):697-703 (2001), and Pizzaro et al., Gene Ther. 8(10):737-745(2001). For replication-deficient viral vectors, such as AAV vectors,complementing cell lines, cell lines transformed with helper viruses, orcell lines transformed with plasmids encoding essential genes, areneeded for replication of the viral vector.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants, or amplifying the gene of interest. Host cells can becultured in serum-containing medium or serum-free medium. Host cells canbe cultured in a serum-free, protein-free, animal component-free medium,including, e.g., a chemically defined medium (e.g., CD OptiCHO™(Invitrogen, #12681)). The cell culture medium can be supplemented, ifdesired, with supplements known to those of skill, such as, e.g., one ormore amino acid(s), such L-glutamine (e.g., 2% v/v 200 mM L-glutamine(Invitrogen, #25031)). The culture conditions, such as temperature, pH,and the like, are those previously used with the host cell selected forexpression, and will be apparent to those skilled in the art and in thereferences cited herein, including, e.g., ANIMAL CELL TECHNOLOGy, Rhielet al., eds., (Kluwer Academic Publishers 1999), Chaubard et al.,Genetic Eng. News 20(18) (2000), Hu et al., ASM News 59:65-68 (1993), Huet al., Biotechnol. Prog. 1:209-215 (1985), Martin et al., Biotechnol.(1987), Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUE,4th ed., (Wiley, 2000), Mather, INTRODUCTION TO CELL AND TISSUE CULTURE:THEORY AND TENCHNIQUE, (Plenum Press, 1998), Freshney, CULTURE OFIMMORTALIZED CELLS, 3rd ed., (John Wiley & Sons, 1996), CELL CULTURE:ESSENTIAL TECHNIQUES, Doyle et al., eds. (John Wiley & Sons 1998), andGENERAL TECHNIQUES OF CELL CULTURE, Harrison et al., eds., (CambridgeUniv. Press 1997).

The nucleic acid also can be contained, replicated, and/or expressed inplant cells. Techniques related to the culture of plant cells aredescribed in, e.g., Payne et al. (1992) PLANT CELL AND TISSUE CULTURE INLIQUID SYSTEMS John Wiley & Sons, Inc. New York, N.Y.; Gamborg andPhillips (eds.) (1995) PLANT CELL, TISSUE AND ORGAN CULTURE: FUNDAMENTALMETHODS SPRINGER LAB MANUAL, Springer-Verlag (Berlin Heidelberg N.Y.)and Plant Molecular Biology (1993) R. R. D. Croy (ed.) Bios ScientificPublishers, Oxford, U.K. ISBN 0 12 198370 6. Cell culture media ingeneral are set forth in Atlas and Parks (eds.) THE HANDBOOK OFMICROBIOLOGICAL MEDIA (1993) CRC Press, Boca Raton, Fla.

For long-term, high-yield production of recombinant proteins, stableexpression systems can be used. For example, cell lines that stablyexpress a polypeptide of the invention can be transduced with expressionvectors comprising viral origins of replication and/or endogenousexpression elements and a selectable marker gene. Following theintroduction of the vector, cells in the cell line may be allowed togrow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells that successfully express the introduced sequences. For example,resistant clumps of stably transformed cells can be proliferated usingtissue culture techniques appropriate to the cell type. Serum-free mediaare readily available (e.g., JRH Biosciences, SAFC Biosciences,Sigma-Aldrich Corporation, worldwide web at sigmaaldrich.com).Serum-free media or conditioned medium (e.g., growth medium previouslyharvested from untransfected or naïve cell cultures) may be preferredfor protein production or cell-banking in some instances.

The invention includes immortalized cells or cell lines comprising oneor more polypeptides (including, e.g., dimeric or monomeric fusionproteins and multimeric polypeptides), conjugates, nucleic acids, orvectors or the invention.

Host cells transformed with an expression vector and/or polynucleotideare optionally cultured under conditions suitable for the expression andrecovery of the encoded protein from cell culture. The polypeptide orfragment thereof produced by such a recombinant cell may be secreted,membrane-bound, or contained intracellularly, depending on the sequenceand/or the vector used. Expression vectors comprising polynucleotidesencoding mature polypeptides of the invention can be designed withsignal sequences that direct secretion of the mature polypeptidesthrough a prokaryotic or eukaryotic cell membrane. Such signal sequencesare typically incorporated into the vector such that the signal sequenceis expressed at the N-terminus of the polypeptide of the invention.Principles related to such signal sequences are discussed elsewhereherein.

Polypeptide Production and Recovery

Following transduction of a suitable host strain and growth of the hoststrain to an appropriate cell density, the selected promoter is inducedby appropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, or other methods,which are well know to those skilled in the art.

As noted, many references are available for the culture and productionof many cells, including cells of bacterial, plant, animal (especiallymammalian) and archebacterial origin. See e.g., Sambrook, Ausubel, andBerger (all supra), as well as Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, Third edition, Wiley-Liss, New York and thereferences cited therein; Doyle and Griffiths (1997) Mammalian CellCulture: Essential Techniques, John Wiley and Sons, NY; Humason (1979)Animal Tissue Techniques, fourth edition W.H. Freeman and Company; andRicciardelli, et al., (1989) In vitro Cell Dev. Biol. 25:1016 1024. Forplant cell culture and regeneration, Payne et al. (1992) Plant Cell andTissue Culture in Liquid Systems, John Wiley & Sons, Inc. New York,N.Y.; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and OrganCulture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin Heidelberg New York) and Plant Molecular Biology (1993) R. R. D.Croy, Ed. Bios Scientific Publishers, Oxford, U.K. ISBN 0 12 198370 6.Cell culture media in general are set forth in Atlas and Parks (eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.Additional information for cell culture is found in available commercialliterature such as the Life Science Research Cell Culture Catalogue(1998) from Sigma-Aldrich, Inc. (St. Louis, Mo.) (“Sigma-LSRCCC”) and,e.g., the Plant Culture Catalogue and supplement (1997) also fromSigma-Aldrich, Inc (St. Louis, Mo.) (“Sigma-PCCS”).

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by any of a number of methods well known inthe art, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g., using any of the tagging systems noted herein),hydroxylapatite chromatography, and lectin chromatography. Proteinrefolding steps can be used, as desired, in completing configuration ofthe mature protein. Finally, high performance liquid chromatography(HPLC) can be employed in the final purification steps. In addition tothe references noted supra, a variety of purification methods are wellknown in the art, including, e.g., those set forth in Sandana (1997)Bioseparation of Proteins, Academic Press, Inc.; and Bollag et al.(1996) Protein Methods, 2.sup.nd Edition Wiley-Liss, NY; Walker (1996)The Protein Protocols Handbook Humana Press, NJ, Harris and Angal (1990)Protein Purification Applications: A Practical Approach IRL Press atOxford, Oxford, England; Harris and Angal Protein Purification Methods:A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993)Protein Purification: Principles and Practice 3.sup.rd Edition SpringerVerlag, NY; Janson and Ryden (1998) Protein Purification Principles,High Resolution Methods and Applications, Second Edition Wiley-VCH, NY;and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ.

One of skill would understand that fusion proteins of the invention(e.g., mutant CTLA-4-Ig fusion protein) can be made by a variety ofmethods described herein, including, e.g., those set forth in Example 1for making LEA29Y-Ig. For example, in place of the LEA29Y-encodingnucleic acid, a nucleic acid sequence encoding a mutant CTLA-4 ECDpolypeptide of the invention (e.g., D3-54 polypeptide) can be clonedinto the IgG2 Fc fusion vector to produce a vector encoding the mutantCTLA-4-Ig fusion protein (e.g., D3-54-IgG2), stable CHO-K1 cellsexpressing such mutant CTLA-4-Ig fusion protein can be made bytransfecting such cells with the mutant CTLA-4-Ig fusionprotein-encoding vector, and the resultant mutant CTLA-4-Ig fusionprotein (e.g., D3-54-IgG2) can be expressed (typically in dimeric form)and purified as described in Example 1.

In Vitro Expression Systems

Cell-free transcription/translation systems can also be employed toproduce recombinant polypeptides of the invention or fragments thereofusing DNAs and/or RNAs of the present invention or fragments thereof.Several such systems are commercially available. A general guide to invitro transcription and translation protocols is found in Tymms (1995)IN VITRO TRANSCRIPTION AND TRANSLATION PROTOCOLS: METHODS IN MOLECULARBIOLOGY, Volume 37, Garland Publishing, New York.

Methods of the Invention

Polypeptides (including, e.g., dimeric and monomeric fusion proteins andmultimeric polypeptides), conjugates, compositions, nucleic acids,vectors, and cells of the invention exhibit a variety of properties andcharacteristics and are believed useful in a variety of applications,including, but not limited to, e.g., in prophylactic or therapeuticmethods for treating a variety of immune system diseases, disorders andconditions in which modulation or regulation of the immune system andimmune system responses may be of benefit. For example, polypeptides,conjugates, compositions, nucleic acids, vectors, and cells of theinvention that have an ability to bind CD80 and/or CD86 or an ECD ofeither or both and/or an ability to inhibit an immune response arebelieved to be useful in prophylactic and/or therapeutic methods forinhibiting or suppressing an immune response in a subject, methods forinhibiting rejection of a tissue, cell, or organ transplant from a donorby a recipient, and other methods described elsewhere herein. Some suchpolypeptides, conjugates, compositions, nucleic acids, vectors, andcells of the invention are expected to be useful in methods for methodof modulating or inhibiting the interaction of T cells expressing CD28and/or CTLA-4 with B7-positive cells.

In one aspect, therapeutic or prophylactic methods of the inventioninvolve administering to a subject an effective amount of at least onesuch polypeptide (including, e.g., fusion protein, multimer, etc.),conjugate, composition, nucleic acid, vector, and/or cell to suppress orinhibit an immune response. In a therapeutic context, the subject istypically one inflicted with an immune system disease, disorder, orcondition, and administration is conducted to prevent furtherprogression of the disease, disorder or condition. For example,administration of a molecule of the invention to a subject sufferingfrom an immune system disease (e.g., autoimmune disease) can result insuppression or inhibition of such immune system attack or biologicalresponses associated therewith. By suppressing this immune system attackon healthy body tissues, the resulting physical symptoms (e.g., pain,joint inflammation, joint swelling or tenderness) resulting from orassociated with such attack on healthy tissues can be decreased oralleviated, and the biological and physical damage resulting from orassociated with the immune system attack can be decreased, retarded, orstopped.

In a prophylactic context, the subject may be one inflicted with,susceptible to, or believed to present an immune system disease,disorder or condition, and administration is typically conducted toprevent progression of the disease, disorder or condition, inhibit oralleviate symptoms, signs, or biological responses associated therewith,prevent bodily damage potentially resulting therefrom, and/or maintainor improve the subject's physical functioning.

In one aspect, the invention provides a method of modulating theinteraction of T cells expressing CD28 and/or CTLA-4 with B7-positivecells, the method comprising contacting B7-positive cells with at leastone of the following in an effective amount to modulate the interactionof B7-positive cells with CD28-positive T cells and/or CTLA-4-positive Tcells: (1) a polypeptide of the invention (e.g., mutant CTLA-4-ECDpolypeptide or dimeric or monomeric mutant CTLA-4-Ig fusion protein);(2) a multimer comprising one of more polypeptides of the invention(e.g., a dimer comprising any two such polypeptides or a tetramercomprising any four such polypeptides); (3) a conjugate comprising atleast one polypeptide of the invention; (4) a nucleic acid of theinvention (e.g., a nucleic acid encoding a polypeptide of theinvention); (5) a vector comprising a nucleic acid of the invention orencoding a polypeptide of the invention; (6) a cell or population ofcells comprising a polypeptide, nucleic acid, conjugate, and/or vectorof the invention; and/or (7) composition of the invention, wherein theinteraction of B7-positive cells with CD28-positive T cells and/orCTLA-4-positive T cells is modulated. Typically, the modulatory effectis an inhibitory effect such that the interaction of B7-positive cellswith CD28-positive T cells and/or CTLA-4-positive T cells is inhibited.In some instances, the B7-positive cells are antigen-presenting cells(APCs). In some such methods, the interaction of B7-2-positive cells(e.g., APCs expressing B7-2 (CD86)) with CD28-positive T cells isinhibited. In some such methods, the interaction of B7-1-positive cells(e.g., APCs expressing B7-1 (CD80)) with CD28-positive T cells isinhibited.

In another aspect, the invention provides a method of inhibiting theinteraction of CD28-positive T cells and/or CTLA-4-positive T cells withB7-positive cells, the method comprising contacting B7-positive cells(e.g., B7-1-positive cells and/or B7-2-positive cells) with at least oneof the following molecules or components of the invention in aneffective amount to inhibit the interaction of CD28-positive T cellsand/or CTLA-4-positive T cells with B7-positive cells: (1) a polypeptideof the invention (e.g., mutant CTLA-4-ECD polypeptide or dimeric ormonomeric mutant CTLA-4-Ig fusion protein); (2) a multimer comprisingone of more polypeptides of the invention (e.g., a dimer comprising anytwo such polypeptides or a tetramer comprising any four suchpolypeptides); (3) a conjugate comprising at least one polypeptide ofthe invention; (4) a nucleic acid of the invention (e.g., a nucleic acidencoding a polypeptide of the invention); (5) a vector comprising anucleic acid of the invention or encoding a polypeptide of theinvention; (6) a cell or population of cells comprising a polypeptide,nucleic acid, conjugate, and/or vector of the invention; and/or (7)composition of the invention, wherein the interaction of CD28-positive Tcells and/or CTLA-4-positive T cells with B7-positive cells isinhibited. In some instances, the B7-positive cells are APCs. In someinstances, the interaction of CD28-positive T cells with hB7-1-positivecells and/or hB7-2-positive cells is inhibited. In some such methods,inhibition of the interaction of CD28-positive T cells withhB7-1-positive cells and/or hB7-2-positive cells results in suppressionor inhibition of one or more of the following: T cell activation orproliferation, cytokine synthesis or production (e.g., production ofTNF-α, IFN-γ, IL-2), induction of various activation markers (e.g.,CD25, IL-2 receptor), inflammation, joint swelling or tenderness, serumlevel of C-reactive protein, anti-collagen antibody production, and/or Tcell-dependent antibody response(s).

In some such methods, at least one such molecule or component of theinvention is administered to a subject in an effective amount to inhibitthe interaction of endogenous CD28-positive T cells with endogenousB7-1-positive cells and/or B7-2-positive cells in the subject. In somesuch methods, the interaction of endogenous CD28-positive T cells withendogenous B7-positive cells expressing B7-2 (CD86) or B7-1 (CD80) isinhibited. In some instances, the B7-positive cells are APCs whichexpress B7-2 or B7-1, and the interaction of B7-2 or B7-1 withCD28-positive T cells is inhibited. In some instances, the interactionof both B7-2 and B7-1 expressed on APCs with CD28-positive T cells isinhibited.

In another aspect, the invention provides a method of suppressing animmune response in vitro or in vivo. The method comprises contacting aB7-positive cells with at least one of the following molecules orcomponents of the invention in effective amount to suppress an immuneresponse: (1) a polypeptide of the invention (e.g., mutant CTLA-4-ECDpolypeptide or dimeric or monomeric mutant CTLA-4-Ig fusion protein);(2) a multimer comprising one of more polypeptides of the invention(e.g., a dimer comprising any two such polypeptides or a tetramercomprising any four such polypeptides); (3) a conjugate comprising atleast one polypeptide of the invention; (4) a nucleic acid of theinvention (e.g., a nucleic acid encoding a polypeptide of theinvention); (5) a vector comprising a nucleic acid of the invention orencoding a polypeptide of the invention; (6) a cell or population ofcells comprising a polypeptide, nucleic acid, conjugate, and/or vectorof the invention; and/or (7) composition of the invention, wherein animmune response is thereby suppressed. One or more immune responses maybe suppressed, including, e.g., T cell response, T cell proliferation oractivation, cytokine synthesis or production, inflammation, jointswelling or tenderness, serum level of C-reactive protein, anti-collagenantibody production, and/or T cell-dependent antibody response(s). Insuch methods comprising contacting a B7-positive cell with a polypeptideof the invention, the polypeptide binds B7-1 (e.g., human B7-1)expressed on B7-positive cells, and/or binds B7-2 (e.g., human B7-2)expressed on B7-positive cells. In some instances, the B7-positive cellsare APCs. In some instances, an immune response is suppressed in vitro,such as in, e.g., an in vitro assay, including those described in detailelsewhere herein (see, e.g., the Examples below). In some instances, animmune response is suppressed in vivo in a subject to whom an effectiveamount to suppress an immune response is administered, such as, e.g., inthe therapeutic or prophylactic treatment methods (e.g., method oftreating rheumatic disease, such as rheumatoid arthritis, or otherautoimmune disease) discussed in detail elsewhere herein.

In another aspect, the invention provides a method of suppressing animmune response in a subject (e.g., mammal, such as a human). The methodcomprises administering to a subject in need thereof with at least oneof the following molecules or components of the invention in atherapeutically or prophylactically effective amount (e.g.,therapeutically or prophylactically effective dose) which suppresses animmune response in the subject: (1) a polypeptide of the invention(e.g., mutant CTLA-4-ECD polypeptide or dimeric or monomeric mutantCTLA-4-Ig fusion protein); (2) a multimer comprising one of morepolypeptides of the invention (e.g., a dimer comprising any two suchpolypeptides or a tetramer comprising any four such polypeptides); (3) aconjugate comprising at least one polypeptide of the invention; (4) anucleic acid of the invention (e.g., a nucleic acid encoding apolypeptide of the invention); (5) a vector comprising a nucleic acid ofthe invention or encoding a polypeptide of the invention; (6) a cell orpopulation of cells comprising a polypeptide, nucleic acid, conjugate,and/or vector of the invention; and/or (7) composition of the invention,wherein an immune response is thereby suppressed in the subject.

In another aspect, the invention provides a method of treating a subjecthaving an immune system disease or disorder modulated by interaction ofendogenous T cells with endogenous cells expressing CD80 and/or CD86.The method comprises administering to a subject in need of suchtreatment a therapeutically effective amount of: (1) a polypeptide ofthe invention (e.g., mutant CTLA-4-ECD polypeptide or dimeric ormonomeric mutant CTLA-4-Ig fusion protein); (2) a multimer comprisingone of more polypeptides of the invention (e.g., a dimer comprising anytwo such polypeptides or a tetramer comprising any four suchpolypeptides); (3) a conjugate comprising at least one polypeptide ofthe invention; (4) a nucleic acid of the invention (e.g., a nucleic acidencoding a polypeptide of the invention); (5) a vector comprising anucleic acid of the invention or encoding a polypeptide of theinvention; (6) a cell or population of cells comprising a polypeptide,nucleic acid, conjugate, and/or vector of the invention; and/or (7)composition of the invention, thereby treating the immune system diseaseor disorder in the subject. If the subject is a human, CD80 is humanCD80, CD86 is human CD86, and CD28 is human CD28. In some such methods,interaction between endogenous T cells expressing CD28 and endogenouscells expressing CD86 and/or endogenous cells expressing CD80 isinhibited.

It is believed that a variety of immune system diseases or disorders,including rheumatic or autoimmune system disease or disorder, may beeffectively treated using one or more of the molecules of the inventiondisclosed herein, such as, e.g., a mutant CTLA-4 ECD polypeptide (e.g.,any of SEQ ID NOS:1-73, such as, e.g., D3-54 (SEQ ID NO:36), D3-69 (SEQID NO:50), or D3-27 (SEQ ID NO:24) mutant CTLA-4 ECD), or a fusionprotein thereof (e.g., D3-54-IgG2 (SEQ ID NO:197 or 211), D3-69-IgG2(SEQ ID NO:199 or 213), D3-29-IgG2 (SEQ ID NO:79 or 210)). The immunesystem disease or disorder may be or involve, e.g., but is not limitedto, Addison's Disease, Allergy, Alopecia Areata, Alzheimer's,Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis,Ankylosing Spondylitis, Antiphospholipid Syndrome (Hughes Syndrome),arthritis, Asthma, Atherosclerosis, Atherosclerotic plaque, autoimmunedisease (e.g., lupus, RA, MS, Graves' disease, etc.), AutoimmuneHemolytic Anemia, Autoimmune Hepatitis, Autoimmune inner ear disease,Autoimmune Lymphoproliferative syndrome, Autoimmune Myocarditis,Autoimmune Oophoritis, Autoimmune Orchitis, Azoospermia, Behcet'sDisease, Berger's Disease, Bullous Pemphigoid, Cardiomyopathy,Cardiovascular disease, Celiac Sprue/Coeliac disease, Chronic FatigueImmune Dysfunction Syndrome (CFIDS), Chronic idiopathic polyneuritis,Chronic Inflammatory Demyelinating, Polyradicalneuropathy (CIPD),Chronic relapsing polyneuropathy (Guillain-Barré syndrome),Churg-Strauss Syndrome (CSS), Cicatricial Pemphigoid, Cold AgglutininDisease (CAD), COPD, CREST syndrome, Crohn's disease, Dermatitis,Herpetiformus, Dermatomyositis, diabetes, Discoid Lupus, Eczema,Epidermolysis bullosa acquisita, Essential Mixed Cryoglobulinemia,Evan's Syndrome, Exopthalmos, Fibromyalgia, Goodpasture's Syndrome,graft-related disease or disorder, Graves'Disease, GVHD, Hashimoto'sThyroiditis, Idiopathic Pulmonary Fibrosis, Idiopathic ThrombocytopeniaPurpura (ITP), IgA Nephropathy, immunoproliferative disease or disorder(e.g., psoriasis), Inflammatory bowel disease (IBD), Insulin DependentDiabetes Mellitus (IDDM), Interstitial lung disease, juvenile diabetes,Juvenile Arthritis, juvenile idiopathic arthritis (JIA), Kawasaki'sDisease, Lambert-Eaton Myasthenic Syndrome, Lichen Planus, lupus, LupusNephritis, Lymphoscytic Lypophisitis, Ménière's Disease, Miller FishSyndrome/acute disseminated encephalomyeloradiculopathy, MixedConnective Tissue Disease, Multiple Sclerosis (MS), muscular rheumatism,Myalgic encephalomyelitis (ME), Myasthenia Gravis, Ocular Inflammation,Pemphigus Foliaceus, Pemphigus Vulgaris, Pernicious Anaemia,Polyarteritis Nodosa, Polychondritis, Polyglandular Syndromes(Whitaker's syndrome), Polymyalgia Rheumatica, Polymyositis, PrimaryAgammaglobulinemia, Primary Biliary Cirrhosis/Autoimmune cholangiopathy,Psoriasis, Psoriatic arthritis, Raynaud's Phenomenon, Reiter'sSyndrome/Reactive arthritis, Restenosis, Rheumatic Fever, rheumaticdisease, Rheumatoid Arthritis, Sarcoidosis, Schmidt's syndrome,Scleroderma, Sjörgen's Syndrome, Solid-organ transplant rejection(kidney, heart, liver, lung, etc.), Stiff-Man Syndrome, Systemic LupusErythematosus (SLE), systemic scleroderma, Takayasu Arteritis, TemporalArteritis/Giant Cell Arteritis, Thyroiditis, Type 1 diabetes, Type 2diabetes, Ulcerative colitis, Uveitis, Vasculitis, Vitiligo, Wegener'sGranulomatosis, and preventing or suppressing an immune responseassociated with rejection of a donor tissue, cell, graft, or organtransplant by a recipient subject. Graft-related diseases or disordersinclude graft versus host disease (GVDH), such as associated with bonemarrow transplantation, and immune disorders resulting from orassociated with rejection of organ, tissue, or cell grafttransplantation (e.g., tissue or cell allografts or xenografts),including, e.g., grafts of skin, muscle, neurons, islets, organs,parenchymal cells of the liver, etc. With regard to a donor tissue,cell, graft or solid organ transplant in a recipient subject, it isbelieved such molecules of the invention disclosed herein (e.g., mutantCTLA-4 ECD polypeptide or mutant CTLA-4-Ig fusion protein) may beeffective in preventing acute rejection of such transplant in therecipient and/or for long-term maintenance therapy to prevent rejectionof such transplant in the recipient (e.g., inhibiting rejection ofinsulin-producing islet cell transplant from a donor in the subjectrecipient suffering from diabetes).

The invention includes any such mutant CTLA-4 ECD polypeptide or mutantCTLA-4-Ig fusion protein of the invention for use in suppressing animmune response associated with at least one of the above immune systemdiseases or disorders. Also provided is the use of any such mutantCTLA-4 ECD polypeptide or mutant CTLA-4-Ig fusion protein of theinvention in the manufacture of a medicament for suppressing an immuneresponse with at least one of the above immune system diseases ordisorders.

An effective amount of a molecule of the invention, such as, e.g., amutant CTLA-4 ECD polypeptide (e.g., D3-54, D3-69, D3-29, D3-56, D3-75)or an Ig fusion protein comprising a mutant CTLA-4 ECD polypeptide ofthe invention (e.g., D3-54-IgG2, D3-69-IgG2, D3-29-IgG2, D3-56-IgG2,D3-75-IgG2, respectively), for suppressing an immune response in asubject or treating an immune system disease or disorder modulated byinteraction of endogenous T cells with endogenous cells expressing CD80and/or CD86 in a subject in the methods described herein may comprisefrom about 0.0001 milligrams per kilogram (mg/kg) weight of the subjectto about 200 milligrams per kilogram (mg/kg) body weight of the subject,such as, e.g., from about 0.001 milligrams per kilogram (mg/kg) bodyweight of the subject to about 100 milligrams per kilogram (mg/kg)weight of the subject, or, e.g., from about 0.001 mg/kg weight of thesubject to at least about 0.005, 0.01, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, or 75mg/kg body weight of the subject. One or more immune responses may besuppressed in the subject, including, e.g., T cell response, T cellactivation or proliferation, cytokine synthesis or production (e.g.,production of TNF-α, IFN-γ, IL-2, etc.), induction of various activationmarkers (e.g., CD25, IL-2 receptor, etc.), synthesis or production ofinflammatory molecules, inflammation, joint swelling, joint tenderness,pain, stiffness, serum levels of C-reactive protein, anti-collagenantibody production, and/or T cell-dependent antibody response(s)). Aneffective amount of a molecule or component of the invention forsuppressing an immune response may be an amount that suppresses animmune response or a symptom or sign thereof by a detectable ormeasurable amount. The immune response may be partially or completelysuppressed. An effective amount for treating an immune system disease ordisorder may be an amount that relieves, lessens, or alleviates at leastone symptom or biological response or effect associated with the diseaseor disorder, prevents progression of the disease or disorder, orimproves physical functioning of the subject.

An effective amount of a molecule or component of the invention formodulating or inhibiting the interaction of T cells expressing CD28and/or CTLA-4 with B7-positive cells may be an amount that modulates orinhibits the binding between B7-positive cells and CD28-positive and/orCTLA-4-positive T cells, respectively. Such binding interaction(s) maybe may be partially or completely modulated or inhibited.

In some such methods, a mutant CTLA-4-Ig fusion protein dimer of theinvention is administered to the subject in a therapeutically orprophylactically effective amount (or dose) sufficient to suppress animmune response, treat an immune system disease or disorder modulated byinteraction of T cells with B7-expressing cells, or modulate or inhibitthe interaction of T cells expressing CD28 and/or CTLA-4 withB7-positive cells. The fusion protein dimer administered is typically asoluble Ig fusion protein dimer. In some such methods, the effectiveamount or dose of the fusion protein dimer of the invention comprisesfrom about 0.001 milligrams per kilogram (mg/kg) body weight of thesubject to about 200 milligrams per kilogram (mg/kg) body weight of thesubject (such as, e.g., a human) or from about 0.001 mg/kg to about 300mg/kg body weight of the subject. For example, the effective amount ordose of the fusion protein dimer may comprise from about 0.001 mg/kgbody weight of the subject to at least about 0.005, 0.01, 0.05, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 25, 30, 40,50, 60, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, or 300 mg/kg bodyweight of the subject (such as, e.g., human, including an adult human).In some instances, the effective amount or dose is from about 0.001milligrams (mg) to about 50 milligrams per kilogram (kg) body weight ofthe subject, including, but not limited to, e.g., from about 0.01 mg/kgto about 100 mg/kg body weight of the subject (e.g., human), from about0.01 mg/kg to about 50 mg/kg body weight of the subject, or from about0.01 mg/kg to about 25 mg/kg weight of the subject; for example, about0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg,0.3 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5mg/kg, 3 mg/kg, 5 mg/kg, about 10 mg/kg, 20 mg/kg, 25 mg/kg, 50 mg/kg,75 mg/kg or 100 mg/kg body weight of the subject (e.g., adult humanpatient) is administered to the subject. In some instances, theeffective amount or dose of the fusion protein dimer is from about 2 to10 mg/kg, about 3 to 10 mg/kg, about 3 to 5 mg/kg, about 5 to 10 mg/kg,0.1 to 5 mg/kg, about 0.05 to 1.0 mg/kg, about 0.05 to 3 mg/kg, about0.05 to 2.0 mg/kg, about 0.05 to 1.0 mg/kg, about 0.1 to 2.0 mg/kg,about 0.1 to 3.0 mg/kg, about 0.1 to 0.5 mg/kg, about 0.1 to 0.8 mg/kg,about 0.1 to 0.6 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about 0.01mg/kg to about 0.1 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about0.01 mg/kg to about 1 mg/kg, about 0.01 to about 5 mg/kg, about 0.01mg/kg to about 3 mg/kg, about 0.05 mg/kg to about 2.5 mg/kg, about 0.1mg/kg to about 1 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.2 to 1mg/kg, about 0.2 to 0.6 mg/kg, about 0.2 to 0.5 mg/kg about 0.3 to 1mg/kg, about 0.3 to 0.6 mg/kg, about 0.3 to 0.5 mg/kg weight of asubject. In some instances, the effective amount or dose is less thanabout 500 mg for a subject weighing less than 60 kg (e.g., less thanabout 100 mg, 75 mg, 50 mg, 25 mg, 12.5 mg. or 10 mg), less than about750 mg for a subject weighing between 60-100 kg (e.g., less than about150 mg, 100 mg, 75 mg, 37.5 mg, or 20 mg), or less than about 1000 mgfor a subject weighing more than 100 kg (e.g., less than about 500 mg,100 mg, 50 mg, 25 mg, or 10 mg).

In another aspect, in some such methods of the invention, a mutantCTLA-4-Ig fusion protein of the invention is administered to the subjectin a therapeutically or prophylactically effective amount or dose thatis, e.g., sufficient to suppress an immune response, treat an immunesystem disease or disorder modulated by interaction of T cells withB7-expressing cells, or modulate or inhibit the interaction of T cellsexpressing CD28 and/or CTLA-4 with B7-positive cells. The effectiveamount or dose of the fusion protein, which is usually a soluble fusionprotein, can comprise from about 0.001 mg/kg to about 300 mg/kg, about0.001 mg/kg to about 200 mg/kg, or about 0.001 mg/kg to about 300 mg/kgbody weight of the subject (e.g., human). In one aspect, the effectiveamount or dose of the fusion protein comprises from about 0.001 mg/kg toat least about 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 2, 3, 4, 5, 10, 25, 30, 40, 50, 60, 75, 80, 90, 100, 125,150, 175, 200, 225, 250, or 300 mg/kg body weight of the subject. Inanother aspect, the effective amount or dose is from about 0.01 mg/kg toabout 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, or from about0.01 mg/kg to about 25 mg/kg weight of the subject. Exemplary doses oramounts include about 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.15 mg/kg,0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 5 mg/kg, about 10 mg/kg, 20 mg/kg,25 mg/kg, 50 mg/kg, 75 mg/kg, and 100 mg/kg body weight of the subject(e.g., adult human). In another aspect, the effective amount or dose ofthe fusion protein is from about 2 to 10 mg/kg, about 3 to 10 mg/kg,about 3 to 5 mg/kg, about 5 to 10 mg/kg, 0.1 to 5 mg/kg, about 0.05 to1.0 mg/kg, about 0.05 to 3 mg/kg, about 0.05 to 2.0 mg/kg, about 0.05 to1.0 mg/kg, about 0.1 to 2.0 mg/kg, about 0.1 to 3.0 mg/kg, about 0.1 to0.5 mg/kg, about 0.1 to 0.8 mg/kg, about 0.1 to 0.6 mg/kg, about 0.01mg/kg to about 0.05 mg/kg, about 0.01 mg/kg to about 0.1 mg/kg, about0.01 mg/kg to about 0.05 mg/kg, about 0.01 mg/kg to about 1 mg/kg, about0.01 to about 5 mg/kg, about 0.01 mg/kg to about 3 mg/kg, about 0.05mg/kg to about 2.5 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.1mg/kg to about 5 mg/kg, about 0.2 to 1 mg/kg, about 0.2 to 0.6 mg/kg,about 0.2 to 0.5 mg/kg about 0.3 to 1 mg/kg, about 0.3 to 0.6 mg/kg,about 0.3 to 0.5 mg/kg weight of a subject. In some aspects, theeffective amount or dose is less than about 500 mg for a subjectweighing less than 60 kg (e.g., less than about 100 mg, 75 mg, 50 mg, 25mg, 12.5 mg. or 10 mg), less than about 750 mg for a subject weighingbetween 60-100 kg (e.g., less than about 150 mg, 100 mg, 75 mg, 37.5 mg,or 20 mg), or less than about 1000 mg for a subject weighing more than100 kg (e.g., less than about 500 mg, 100 mg, 50 mg, 25 mg, or 10 mg).

The effective amount or dose of a nucleic acid, vector, composition,and/or cell of the invention sufficient to similarly suppress an immuneresponse or modulate, treat an immune system disease or disordermodulated by interaction of T cells with B7-expressing cells, ormodulate or inhibit the interaction of T cells expressing CD28 and/orCTLA-4 with B7-positive cells can be determined. For example, if avector encoding such a fusion protein dimer of the invention is to beadministered to the subject, one skilled in the art can readilydetermine the amount of the vector to be administered such that adesired therapeutically or prophylactically effective amount of thefusion protein dimer is likely produced in the subject.

Exemplary fusion protein dimers of the invention include any of thosedescribed in detail above and herein, including, e.g., a fusion proteindimer comprising two identical fusion protein monomers, wherein eachfusion protein monomer comprises a mutant CTLA-4 ECD polypeptide of theinvention fused at its C-terminus to the N-terminus of an Ig Fcpolypeptide (e.g., IgG2 Fc, IgG1, IgG4 or mutant Ig Fc polypeptide whichreduces effector function). An exemplary mutant CTLA-4 ECD polypeptideis one comprising a polypeptide sequence selected from the groupconsisting of SEQ ID NOS:1-73. An exemplary fusion protein dimer is onecomprising two fusion protein monomers, wherein each fusion proteinmonomer comprises a polypeptide sequence selected from the groupconsisting of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222.Typically, the two monomeric fusion proteins in a dimeric fusion proteinare covalently linked together via at least one disulfide bond formedbetween cysteine residue(s) present in each monomer.

In any of the methods described above, the molecule or component of theinvention (e.g., polypeptide (including, e.g., dimeric or monomericfusion protein or polypeptide multimer), conjugate, nucleic acid,vector, composition, and/or cell of the invention) may be administeredto the subject as a composition. The composition typically comprises atleast one such molecule or component and an excipient, carrier, ordiluent. The composition may comprise a pharmaceutical compositioncomprising at least one such molecule or component and apharmaceutically acceptable excipient, carrier, or diluent (e.g., PBS).The pH of compositions of the invention typically ranges from about pH6.0 to about pH 9.0, including, e.g., from about pH 6.5 to about pH 8.5,usually from about pH 7.0 to about pH 8.0. In one aspect, the pH ofcompositions of the invention typically ranges from about pH 3 to aboutpH 10, from about pH 4 to about pH 10, from about pH 5 to about pH 9,from about pH 6 to about pH 9, from about pH 5.5 to about pH 8.5, fromabout pH 6.0 to about pH 6.7, from about pH 6.0 to about pH 6.5, fromabout pH 6.2 to about pH 8.7, from about pH 6.5 to about pH 7.5, fromabout pH 6.2 to about pH 7.0, from about pH 6.3 to about pH 6.8, fromabout pH 6.4 to about pH 6.8, and about pH 7.0 to about pH 7.4. In oneaspect, a composition comprising at least one such molecule or componentof the invention, such as, e.g., a mutant CTLA-4-Ig fusion protein, hasa pH of pH 5.5, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6,pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5,pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4,pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9.0, pH 9.1, pH 9.2, pH 9.3,pH 9.4, pH 9.5, pH 9.6, pH 9.7, pH 9.8, pH, 9.9, or pH 10.0. Somecompositions of the invention include one or more salts (e.g., sodiumchloride, sodium phosphate, calcium chloride, and the like), one or morebuffers (e.g., HEPES, sodium citrate, sodium phosphate (e.g.,Na₂HPO₄/Na₃PO₄), succinate, tartrate, fumarate, gluconate, oxalate,lactate, acetate, tris(hydroxymethyl)aminomethane (Tris), and the like),one, two three, four, five, or more saccharides or sugars (e.g.,sucrose, mannose, maltose, trehalose, dextrose, and the like), and/orone, two, three, four or more polyalcohols or sugar alcohols (e.g.,mannitol, sorbitol, glycol, glycerol, arabitol, erythritol, xylitol,ribitol, lactitol, and the like). One, two three, four, five, or moremonosaccharides, disaccharides and/or polysaccharides can be included inthe composition. The composition of the invention may comprise anyconcentration of such molecule or component effective to suppress animmune response when administered to the subject. For example, in somesuch methods (including, e.g., methods in which immunosuppression isdesirable, such as, but not limited, to, e.g., treatment of rheumatoidarthritis or similar immune disorders, or for inhibiting rejection of atissue, cell, graft, or organ transplant from a donor by a recipientsubject), a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier, excipient, or diluent and a fusion protein dimer ofthe invention is administered to the subject (e.g., parentally,subcutaneously, intravenously, intramuscularly, etc.), wherein thepharmaceutical composition comprises a fusion protein dimer of theinvention at a concentration of from about 0.001 mg/ml to about 200mg/ml, about 0.001 mg/ml to about 300 mg/ml, about 0.01 mg/ml to about200 mg/ml, about 0.01 mg/ml to about 250 mg/ml, about 0.1 mg/ml to about200 mg/ml, about 0.001 mg/ml to about 100 mg/ml, about 0.001 mg/ml toabout 90 mg/ml, about 0.01 mg/ml to about 90 mg/ml, about 0.01 mg/ml toabout 75 mg/ml, about 0.1 to about 80 mg/ml, about 0.1 to about 75mg/ml, about 0.1 to about 60 mg/ml, about 0.1 to about 50 mg/ml, about0.1 to about 40 mg/ml, about 0.1 to about 30 mg/ml, about 1 to about 90mg/ml, about 1 to about 80 mg/ml, about 1 to about 75 mg/ml, about 1 toabout 60 mg/ml, about 1 to about 50 mg/ml, about 1 to about 40 mg/ml,about 1 to about 30 mg/ml, about 1 to about 20 mg/ml, about 1 to about10 mg/ml, about 1 to about 5 mg/ml, about 5 to about 90 mg/ml, about 5to about 80 mg/ml, about 5 to about 75 mg/ml, about 5 to about 60 mg/ml,about 5 to about 50 mg/ml, about 5 to about 40 mg/ml, about 5 to about30 mg/ml, about 5 to about 20 mg/ml, about 5 to about 10 mg/ml, about 1to about 5 mg/ml, about 10 to about 75 mg/ml, about 25 mg/ml to about 75mg/ml, about 30 mg/ml to about 60 mg/ml, about 25 to about 50 mg/ml,about 50 mg/ml to about 100 mg/ml, including, e.g., about 1 mg/ml, 5mg/ml, 10 mg/ml, about 15 mg/ml, about 25 mg/ml, about 30 mg/ml, about40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80mg/ml, about 90 mg/ml, or 100 mg/ml. Other concentrations arecontemplated. In some methods of the invention described herein,including some therapeutic or prophylactic methods, a volume of any suchcomposition (e.g., pharmaceutical composition) comprising a fusionprotein of the invention in a range of about 0.01 milliliter (ml) toabout 10 ml, about 0.01 ml to about 5 ml, about 0.1 ml to about 5 ml,about 0.5 ml to about 2 ml, about 1 ml to about 2 ml, including, e.g., avolume of 0.01 ml, 0.025 ml, 0.05 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml,0.5 ml, 0.75 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 20 ml, 25 ml, 50ml, 75 ml, 100 ml, 200 ml, 250 ml, 300 ml, 500 ml, 1000 ml, etc. isadministered to a subject via a single i.v., s.c., i.m., or i.p.injection. Further details of exemplary compositions of the inventionare discussed elsewhere herein.

The effective amount or dose of a molecule of the invention that isadministered to a particular subject may vary depending upon, e.g., thedisease, disorder, or condition being treated, the potency of theparticular mutant CTLA-4 molecule of the invention (i.e., its efficacy)(e.g., a mutant CTLA-4-Ig fusion protein dimer of the invention) to beadministered, the mode of administration of the molecule, and thesubject's individual ability to tolerate a specific amount of theparticular molecule. For example, in a method for suppressing an immuneresponse in a subject having rheumatoid arthritis (RA) or a method fortreating RA, the effective amount or dose of a mutant CTLA-4-Ig dimer ofthe invention (e.g., D3-29-IGg2, D3-54-IgG2, D3-56-IgG2, D3-69-IgG2,D3-75-IgG2, etc.) to be administered to the subject can be determinedbased on a variety of factors, including the potency of the mutantCTLA-4-Ig dimer, the mode of administration of the dimer, and/or theseverity of the subject's symptoms or signs of rheumatoid arthritis. Inone aspect, an effective amount or dose of a particular mutant CTLA-4-Igdimer of the invention can be determined by comparing the potency ofsuch mutant CTLA-4-Ig dimer with that of the Orencia® dimer. Doses ofthe Orencia® dimer effective for treating rheumatoid arthritis andrelated disorders are known in the art. For example, the Orencia® dimeris typically administered intravenously to a human suffering fromrheumatoid arthritis in a dose of about 10 mg Orencia® per kilogram (kg)body weight of the human. A mutant CTLA-4-Ig dimer of the invention thatis about “X” times more potent than Orencia® can be administered (e.g.,intravenously, subcutaneously, or in another manner described herein) toa human suffering from rheumatoid arthritis in a dose that is about “X”times less than the Orencia® dimer dose to achieve a therapeutic effect(e.g., suppressing an immune response) that is approximately equivalentto that of the Orencia® dimer. If a greater therapeutic effect isdesired, a proportionally increased amount or dose of the mutantCTLA-4-Ig dimer can be readily determined and administered to the human.

In any of the methods described herein, the molecule or component of theinvention (e.g., a polypeptide (including, e.g., dimeric or monomericfusion protein or polypeptide multimer), conjugate, nucleic acid,vector, composition, and/or cell of the invention) can be administeredparentally, subcutaneously, or intravenously, or as described elsewhereherein. The molecule or component of the invention may be administeredin a therapeutically effective amount one, two, three or four times permonth, two times per week, biweekly (every two weeks), or bimonthly(every two months). Administration may last for a period of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 months or longer (e.g., one, two, three,four or more years, including for the life of the subject).

Any of the methods described herein may further comprise administeringto the subject an effective amount of at least one additionaltherapeutic or immunosuppressive agent or compound. Thus, for example,the invention provides a method of suppressing an immune responsecomprising administering to a subject in need thereof (1) an effectiveamount of at least one first immunosuppressive agent, wherein each suchfirst immunosuppressive agent is a polypeptide, nucleic acid, vector,composition, and/or cell of the invention, and (2) an effective amountof at least one second immunosuppressive agent, wherein an immuneresponse in the subject is suppressed.

A variety of additional therapeutic or immunosuppressive agents (thatare not molecules of the invention) may be used or administered inconjunction with a molecule of the invention (e.g., polypeptide, nucleicacid, vector, composition, and/or cell of the invention). Such agentsinclude, e.g., a disease-modifying anti-rheumatic drug (DMARD) (such as,e.g., methotrexate (MTX), cytokine antagonist (e.g., IL-2 or IL-6antagonist), steroidal compound (e.g., corticosteroid, glucocosteroid,e.g., prednisone or methylprednisone), non-steroidal compound, sodium ormagnesium salicylate, ibuprofen, acetylsalicylic acid, acetaminophen,antibody, biological agent that blocks synthesis of an productionanti-inflammatory cytokine, Raptiva® efalizumab, anti-inflammatory agentor compound, and non-steroidal anti-inflammatory drug (NSAID). Suchadditional therapeutic or immunosuppressive agent can be administered tothe subject in a pharmaceutical composition comprising the additionalagent and a pharmaceutically acceptable excipient or carrier. Theeffective amount or dose of the agent to be administered will dependupon the specific agent. Some such agents are currently used inimmunosuppressive therapies and appropriate dosages can be determined bybased upon the disease, disorder or condition being treated and thesubject's ability to tolerate specific amounts or doses, and the agent'simmunosuppressive effectiveness. Exemplary doses for immunosuppressiveagents described above which are not molecules of the invention areknown. The additional immunosuppressive agent that is not a molecule ofthe invention can be administered simultaneously with or before or afteradministration of the molecule of the invention (e.g., mutant CTLA-4-Igfusion protein).

A treatment regimen, including, e.g., dose, schedule of administration,method of administration (e.g., intravenous injection, subcutaneousinjection, etc.) and pharmaceutical composition comprising at least onesuch molecule or component of the invention may vary depending upon thedisease, disorder or condition to be treated. One or more such moleculesor components of the invention may be administered to a subject; eachsuch molecule or component need not be administered in the samepharmaceutical formulation, by the same administration methods, in thesame amount, or by the same dosing frequency schedule.

In some such methods, for example, about 1 ml of a pharmaceuticalcomposition comprising a pharmaceutically acceptable excipient, carrier,or diluent and a concentration of a fusion protein dimer of theinvention of about 50 mg/ml is administered subcutaneously to a subject(e.g., adult human) in need of immunosuppression (e.g., a subjectsuffering from rheumatoid arthritis). Such initial dose is 50 mg offusion protein dimer. For a subject having a body weight of 100 kg, thisinitial dose corresponds to 0.5 mg fusion protein dimer per kg bodyweight of the subject. A second of the same amount is administeredsubcutaneously at one or two weeks after the first dose. Further dosesare administered subcutaneously every week, biweekly, or once per month,or more or less frequently as necessary. Such compositions andadministration formats are believed useful, for example, for treating ahuman suffering from rheumatoid arthritis or another immune disorder inwhich immunosuppression is desirable or for inhibiting rejection of atissue, cell, graft, or organ transplant from a human donor by a humanrecipient.

Methods of Treating Rheumatoid Arthritis

Rheumatoid arthritis is one of the most common systemic inflammatoryautoimmune diseases and is estimated to affect 1-2% of the adultpopulation. See, e.g., Dipiro, J. T., Rheumatoid arthritis, inPHARMACOTHERAPY: A PATHOPHYSIOLOGIC APPROACH, 1671-1682 (Talbert, R. T.et al. eds., McGraw-Hill, New York, 6^(th) ed. 2005). The disease ischaracterized by synovial membrane hyperplasia and infiltration ofinflammatory cells, including activated T cells. Activated T cells playa pivotal role in the progression of rheumatoid arthritis by stimulatinga variety of cell types to produce proinflammatory cytokines, such asIL-1, IL-6, and TNF-alpha, autoantibodies, and matrix metalloproteinases(Hoffman, R. W., Front. Biosci. 6:1369-1378 (2001); Choy, E. K. et al.,N. Engl. J. Med 344:907-916 (2001)). The strong contribution of T cellsto the progression of rheumatoid arthritis makes T cell activation arational target for therapeutic intervention. Such inflammatorymolecules are believed to cause the inflammatory response, tissue damage(e.g., joint damage), and pain associated with rheumatoid arthritis.

Co-stimulation of T cells mediated by interactions between CD28 receptorand CD80 and/or CD86 ligand(s) is essential for the activation of most Tcells (Riley, J. L. et al., Blood 105:13-21 (2005)). Therapeutic orprophylactic agents that antagonize the CD80/CD86-CD28 co-stimulationpathway, such as the Orencia® (Abatacept) fusion protein, which is asoluble dimeric hCTLA-4-Ig fusion protein, have been shown to beclinically effective in the treatment of rheumatoid arthritis (Kremer,J. M. et al., Ann. Intern. Med. 144:865-876 (2006); Genovese, M. C. etal., N. Engl. J. Med. 353:1114-1123 (2005)). Abatacept is believed toexert immunosuppressive function by binding to CD80 and/or CD86 ligandson antigen-presenting cells when administered to a subject (e.g., adulthuman) in vivo in a therapeutically or prophylactically effectiveamount, thus preventing the interaction of either or both of theseligands with the CD28 receptor on T cells.

Abatacept is presently approved to treat adult human patients withmoderately to severely active RA who have had an inadequate response toone or more DMARDs, such as methotrexate or TNF antagonists. Abataceptis administered to an adult RA patient at a dose of 10 mg/kg body weightof the subject by intravenous infusion. Following the first dose, secondand third doses of 10 mg/kg of the fusion protein are administered tothe subject at two and four weeks, respectively, after the first dose.Subsequent doses are administered every four weeks (i.e., once permonth). Intravenous infusion of Abatacept is believed necessary todeliver the high dose level required to obtain desirable efficacy inrheumatoid arthritis therapy.

Other current therapies for rheumatoid arthritis include theadministration of non-specific immunosuppressive agents, such asmethotrexate, and steroidal and non-steroidal anti-inflammatory drugs.Additionally, biologic agents are approved that target specificpro-inflammatory cytokines, such as TNF-α (e.g., Remicade® infliximab,Enbrel® entaercept, Humira® adalimumab) and IL-1 (e.g., Kineret®anakinra). However, many of these therapies have significant sideeffects—some of which are toxic—particularly when administered over along time period.

Despite the availability of various therapies, significant unmet needexists for the treatment of RA. For example, 60% of human RA patientswho have failed previous DMARD treatment and 80% of human RA patientswho have failed previous anti-TNF therapy did not achieve an ACR50scores after treatment with Orencia for 6 months (Kremer J. M. et al.,Ann. Intern. Med. 144:865-876 (2006); Genovese, M. C. et al., N. Engl.J. Med. 353:1114-11 (2005)). Dose response studies using Abatacept andBelatacept (LEA29Y-Ig) fusion protein in the treatment of RA in adultsindicated that efficacy was dose-dependent and was not saturated at thehighest dose levels tested (Kremer, J. M. et al., N. Engl. J. Med.349:1907-1915 (2003); Moreland, L. W. et al., Arthrit. Rheum.46:1470-1479 (2002)).

A soluble dimeric mutant CTLA-4-Ig of the invention having a higherbinding avidity to hCD80 and/or hCD86 than Abatacept is expected to beable to exert more potent immunosuppressive effects than Abatacept whenadministered to a subject with RA. Such a mutant CTLA-4-Ig binds asimilar number of CD80 and/or CD86 ligands at a lower concentration thanAbatacept.

A mutant CTLA-4-Ig with a higher binding avidity to CD80 or CD86 andslower dissociation rate from CD80 or CD86, respectively, has a longerresidence time on such ligand. This longer residence time is expected tobe associated with higher efficacy in vivo. It is believed that such amutant CTLA-4-Ig may be effective in therapeutically or prophylacticallytreating a subject with RA at a dose that is lower than the Abatacept.That is, it is believed that such a mutant CTLA-4-Ig may achieve adegree of efficacy equivalent to that of Abatacept when administered tothe RA subject at a dose that is less than that of the Abatacept dose of10 mg/kg body weight of the subject. The invention provides solubledimeric mutant CTLA-4-Ig fusion proteins of varied binding avidities tohCD80 and/or hCD86. Soluble dimeric mutant CTLA-4-Ig fusion proteinsthat have substantially higher binding avidities to hCD86 than Abataceptmay a degree of efficacy equivalent to that of Abatacept whenadministered to the RA subject at a dose that is substantially less thanthat of Abatacept. Administration of a lower dose of such a mutantCTLA-4-Ig may allow a more convenient method of administration (e.g.,subcutaneous injection) to be used than is currently used foradministration of Abatacept (intravenous injection).

It is also believed that a soluble mutant CTLA-4-Ig fusion protein ofthe invention with a higher immunosuppressive potency than Abatacept orBelatacept fusion protein would enable a higher level of efficacy to beobtained in the treatment of RA patients. A more immunosuppressivemutant CTLA-4-Ig is expected to be able to alleviate symptoms associatedwith RA and inhibit progressive of the deleterious physical effects ofRA more effectively than Abatacept. Such a mutant CTLA-4-Ig can beformulated in a pharmaceutically acceptable diluent, excipient, orcarrier (e.g., PBS) at a concentration ranging from of 0.1-200 mg/ml.Treatment of a subject with RA can be accomplished by administering tothe subject a therapeutically or prophylactically effective amount(dose) of the mutant CTLA-4-Ig by subcutaneous injection or intravenousinfusion at an appropriately determined dosing frequency (e.g., initialdose following by one dose 2 to 4 times per month, one dose per month,or one dose every two months). The dose would depend upon the severityof the subject's disease or symptoms. For example, an amount or dose ofa mutant CTLA-4-Ig of no more than about 10 mg/kg (including e.g., about1 mg/kg, 0.5 mg/kg, 0.25 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, or 9 mg/kg) body weight of the subjectmay be administered. A more immunosuppressive mutant CTLA-4-Ig may allowfor a less frequent dosing schedule (e.g., once very two months) thanthe dosing schedule typically used with Abatacept. Alternatively, anamount or dose of a mutant CTLA-4-Ig greater than about 10 mg/kg weightof the subject (e.g., from about 10 mg/kg to about 100 mg/kg, from about10 mg/kg to about 25 mg/kg, about 10 mg/kg to about 50 mg/kg, about 10mg/kg to about 75 mg/kg, etc., including, e.g., about 15 mg/kg, 20mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 g/kg, 70 mg/kg, 75 mg/kg, 80mg/kg, 90 mg/kg, 100 mg/kg) may be administered to a subject with RA ifthe subject's disease condition and/or symptoms warrants such amount ordose.

The effective amount or dose of a mutant CTLA-4-Ig dimer of theinvention for treating RA in a human suffering therefrom can bedetermined based on various factors, such as the potency of the mutantCTLA-4-Ig dimer, the mode of administration of the dimer, and/or theseverity of the subject's symptoms or signs of rheumatoid arthritis. Forexample, an effective amount or dose of a mutant CTLA-4-Ig dimer of theinvention can be ascertained by comparing the potency of such dimer tothat of the Orencia® dimer and determining the amount or dose of themutant CTLA-4-Ig dimer that would yield the desired immunosuppressiveeffect compared to Orencia® (e.g., an improved or approximatelyequivalent effect) based on amount or dose of Orencia® that wouldtypically be administered to a human subject exhibiting similar symptomsor signs of RA.

In one embodiment, for example, the invention provides a method oftreating rheumatoid arthritis in a subject in need of such treatment,the method comprising administering to the subject an effective amountof a soluble dimeric mutant CTLA-4-Ig fusion protein of the inventionby, e.g., intravenous or subcutaneous injection. The effective amount ordose may comprise from about 0.001 milligrams (mg) to about 10milligrams per kilogram (kg) body weight of the subject, including, butnot limited to, e.g., from about 0.01 mg/kg to about 10 mg/kg weight ofthe subject, from about 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.25 mg/kg,0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8mg/kg, 9 mg/kg, or 10 mg/kg body weight of the adult human patient isadministered to the subject. In some instances, the effective amount ordose is from about 2 to 10 mg/kg, about 3 to 10 mg/kg, about 3 to 5mg/kg, about 5 to 10 mg/kg, 0.1 to 5 mg/kg weight, about 0.05 to 1.0mg/kg, about 0.05 to 3 mg/kg weight, about 0.05 to 2.0 mg/kg, about 0.05to 1.0 mg/kg, about 0.05 to 2.0 mg/kg, about 0.1 to 2.0 mg/kg, about 0.1to 3.0 mg/kg, about 0.1 to 0.5 mg/kg, about 0.1 to 0.8 mg/kg, about 0.1to 0.6 mg/kg, about 0.2 to 1 mg/kg, about 0.2 to 0.6 mg/kg, about 0.2 to0.5 mg/kg about 0.3 to 1 mg/kg, about 0.3 to 0.6 mg/kg, about 0.3 to 0.5mg/kg weight of a subject. In some instances, the effective amount ordose is less than about 500 mg for a subject weighing less than 60 kg(e.g., less than about 100 mg, 75 mg, 50 mg, 25 mg, or 12.5 mg), lessthan about 750 mg for a subject weighing between 60-100 kg (e.g., lessthan about 150 mg, 100 mg, 75 mg, 37.5 mg, or 20 mg), or less than about1000 mg for a subject weighing more than 100 kg (e.g., less than about500 mg, 100 mg, 50 mg, 25 mg, or 10 mg). Following the first dose,subsequent equivalent doses are administered at intervals of 1, 2, 4, 8,10, 12, 14, or 16 weeks. Subsequent dosing frequency can be determinedas needed.

Such a mutant CTLA-4-Ig fusion may be formulated with a pharmaceuticallyacceptable excipient, carrier, or diluent to produce a pharmaceuticalcomposition suitable for administration to a subject (e.g., mammal,including a human). The concentration of the fusion protein in thecomposition may range from about 0.01 mg/ml to about 300 mg/ml or fromabout 0.01 mg/ml to about 200 mg/ml, including, but not limited to,e.g., from about 0.1 mg/ml to about 300 mg/ml, from about 0.1 mg/ml toabout 200 mg/ml, about 0.1 mg/ml to about 100 mg/ml, about 0.5 mg/ml toabout 100 mg/ml, about 0.5 mg/ml to about 50 mg/ml, about 1 to about 100mg/ml, about 1 to about 75 mg/ml, about 5 to about 75 mg/ml, about 10 toabout 75 mg/ml, about 10 to about 60 mg/ml, about 25 to about 60 mg/ml,about 30 to about 60 mg/ml, about 25 to about 50 mg/ml, about 40 toabout 50 mg/ml, about 25 mg/ml, or about 50 mg/ml. Other compositions,including those discussed above and below, are also contemplated.

Such treatment is expected to reduce one or more signs and/or symptomsassociated with rheumatoid arthritis, such as, e.g., inflammation, jointtenderness, joint swelling, pain, and stiffness, in the subject. Suchtreatment may reduce the further progression of the disease in thepatient. For example, such treatment may reduce the progression ofstructural damage in the patient. Such treatment may improve physicalfunctioning of the subject.

Methods of Inhibiting Tissue, Cell, Graft, or Organ TransplantationRejection

In another aspect, the invention provides a method of inhibitingrejection of, or suppressing an immune response associated with, atissue, cell, skin graft, or organ transplant from a donor by arecipient subject, the method comprising administering to the recipientsubject a therapeutically effective amount of one or more of thefollowing: (1) a polypeptide of the invention (e.g., mutant CTLA-4-ECDpolypeptide or dimeric or monomeric mutant CTLA-4-Ig fusion protein);(2) a multimer comprising one of more polypeptides of the invention(e.g., a dimer comprising any two such polypeptides or a tetramercomprising any four such polypeptides); (3) a conjugate comprising atleast one polypeptide of the invention; (4) a nucleic acid of theinvention (e.g., a nucleic acid encoding a polypeptide of theinvention); (5) a vector comprising a nucleic acid of the invention orencoding a polypeptide of the invention; (6) a cell or population ofcells comprising a polypeptide, nucleic acid, conjugate, and/or vectorof the invention; and/or (7) a composition of the invention, therebyinhibiting rejection of the tissue, cell, skin graft, or organtransplant by the recipient subject. The donor and recipient may be thesame species or different species. The donor or recipient may be amammal, such as a human, non-human primate (e.g., monkey, gorilla),sheep, cat, dog, pig, cow, horse, etc. In some such methods, thepolypeptide, conjugate, vector, and/or cell of the invention isadministered to the recipient subject prior to, simultaneously with, orafter tissue, cell, skin graft, or organ transplantation. The effectiveamount typically comprises from about 0.001 mg/kg weight of the subjectto about 200 mg/kg body weight of the subject. In some such methods, forexample, the effective amount comprises from about 0.001 milligrams perkilogram (mg/kg) weight of the subject to at least about 0.005, 0.01,0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10,25, 50, 75, 100, 125, 150, 175, 200, 225, 250, or 300 milligrams perkilogram (mg/kg) body weight of the subject. In some such methods, theeffective amount comprises from about 0.001 milligrams per kilogram(mg/kg) weight of the subject to at least about 0.005, 0.01, 0.05, 0.1,0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 50, or 75 milligramsper kilogram (mg/kg) body weight of the subject. The polypeptide,conjugate, nucleic acid, vector, and/or cell of the invention may beadministered to the recipient subject during, prior to, or immediatelyafter the transplantation. Alternatively or additionally, such moleculeof the invention may be administered one or more hours aftertransplantation, on the day following transplantation, and/or dailythereafter, or at least once per week, at least once every two weeks, orat least one per month after transplantation, as necessary, for up to12, 24, or 36 or more months or longer as needed. The organ transplantmay involve any organ, such as, e.g., a kidney, liver, heart, or lung.

The effective amount or dose of a mutant CTLA-4 molecule of theinvention (e.g., mutant CTLA-4-Ig fusion protein dimer) to beadministered to an organ, tissue, or cell transplant recipient subjectso as to inhibit transplant rejection (or suppress an immune responseassociated with such transplant) is typically determined based on thepotency of such molecule, mode of administration, the type oftransplantation (e.g., cell, tissue, organ), the subject's history,and/or the severity of the transplant recipient subject's symptoms orsigns of an immune response(s) suggestive of transplant rejection. Forexample, an effective amount or dose of a mutant CTLA-4-Ig dimer of theinvention (e.g., D3-29-IGg2, D3-54-IgG2, D3-56-IgG2, D3-69-IgG2,D3-75-IgG2 dimer, etc.) can be determined by comparing the potency ofsuch dimer with that of the Belatacept dimer. Effective doses ofBelatacept useful for preventing or suppressing an immune responseassociated with kidney/renal transplantation are known. For example,Belatacept is administered by intravenous infusion to a human followingkidney transplantation in the human from a kidney donor in an amount ordose of about 5 mg or 10 mg per kilogram body weight of the human permonth. A mutant CTLA-4-Ig dimer of the invention that is about “X” timesmore potent than Belatacept can be administered (e.g., intravenously,subcutaneously, or in another manner described herein) to a human whohas had kidney transplant in an amount or dose that is about “X” timesless than the Belatacept dose to achieve a therapeutic effect (e.g.,suppressing an immune response) approximately equivalent to that ofBelatacept. If a greater therapeutic effect is desired, a proportionallyincreased amount or dose of the CTLA-4-Ig fusion protein dimer of theinvention can be determined and administered.

In another aspect, the invention provides a method of treating tissue,cell, or organ transplant rejection (e.g., solid organ transplantrejection (e.g., kidney, liver, lung, heart, etc.)) in a subject whoreceives such tissue, cell, or organ from a donor, the method comprisingadministering to the recipient a therapeutically effective amount of atleast one polypeptide, conjugate, nucleic acid, vector, and/or cell ofthe invention, thereby inhibiting rejection of the donor tissue, cell,or organ transplant by the recipient subject. The polypeptide,conjugate, nucleic acid, vector, and/or cell of the invention can beadministered to the subject prior to, simultaneously with, or aftercell, tissue or organ transplantation.

In one aspect, the invention provides a method of inhibiting rejectionof islet cell transplantation from a donor in a recipient subject inneed thereof, the method comprising administering to the subject aneffective amount or dose of a mutant CTLA-4 molecule of the invention(e.g., mutant CTLA-4-Ig fusion protein) prior to, simultaneously with,or after transplantation of islet cell(s) from the pancreas of a donorinto the subject. The subject (e.g., human) typically suffers fromdiabetes (e.g., IDDM) and such method is useful in treating a subjectdiagnosed with or suffering from diabetes. Islet transplantationprocedures are known in the art. Typically, islets are removed from thepancreas of a deceased organ donor, purified and processed, andimplanted into a recipient subject suffering from diabetes. Aftertransplantation, the beta cells in the islets begin to make and releaseinsulin, thereby reducing the recipient subject's need for insulin.

In such methods of inhibiting transplant rejection, the mutant CTLA-4molecule of the invention (e.g., mutant CTLA-4-Ig) can be formulatedwith a pharmaceutically acceptable excipient, carrier, or diluent toproduce a pharmaceutical composition suitable for administration to asubject (e.g., mammal, including a human). Some such methods compriseadministration of a pharmaceutical composition comprising apharmaceutically acceptable excipient, carrier, or diluent and a mutantCTLA-4-Ig dimer of the invention having a concentration of from about0.01 mg/ml to about 300 mg/ml or about 0.01 mg/ml to about 200 mg/ml,including, but not limited to, e.g., from about 0.1 mg/ml to about 300mg/ml, from about 0.1 mg/ml to about 200 mg/ml, about 0.1 mg/ml to about100 mg/ml, about 0.5 mg/ml to about 100 mg/ml, about 0.5 mg/ml to about50 mg/ml, about 1 to about 100 mg/ml, about 1 to about 75 mg/ml, about 5to about 75 mg/ml, about 10 to about 75 mg/ml, about 10 to about 60mg/ml, about 25 to about 60 mg/ml, about 30 to about 60 mg/ml, about 25to about 50 mg/ml, about 40 to about 50 mg/ml, about 25 mg/ml, or about50 mg/ml. Other compositions, including those discussed above and below,are also contemplated.

Methods of Inhibiting an Immune Response

In another aspect, the invention includes the use of a polypeptide(including, e.g., a dimeric or monomeric fusion protein or multimericpolypeptide), conjugate, nucleic acid, vector, or cell of the inventionfor the manufacture of a medicament for inhibiting or suppressing animmune response in a mammal (e.g., human or non-human primate). Immuneresponses that may be suppressed include, e.g., T cell activation orproliferation, cytokine synthesis or production, induction of activationmarkers, synthesis or production of inflammatory molecules,inflammation, anti-collagen Ab production, T cell-dependent Ab response.

The invention also includes the use of a polypeptide (including, e.g., adimeric or monomeric fusion protein or multimeric polypeptide),conjugate, nucleic acid, vector, or cell of the invention for themanufacture of a medicament for the treatment of an immune systemdisease or disorder. The immune system disease or disorder may be onethat is mediated by interaction of T cells with CD80-positive cellsand/or CD86-positive cells in a mammal. The immune system disease ordisorder may be an immune system disease or disease, such as a rheumaticdisease or disorder or an autoimmune disease or autoimmune disorder.Such immune system disease or disorder may be or involve, e.g., but isnot limited to, Addison's Disease, Allergy, Alopecia Areata,Alzheimer's, Antineutrophil cytoplasmic antibodies (ANCA)-associatedvasculitis, Ankylosing Spondylitis, Antiphospholipid Syndrome (HughesSyndrome), arthritis, Asthma, Atherosclerosis, Atherosclerotic plaque,autoimmune disease (e.g., lupus, RA, MS, Graves' disease, etc.),Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune inner eardisease, Autoimmune Lymphoproliferative syndrome, AutoimmuneMyocarditis, Autoimmune Oophoritis, Autoimmune Orchitis, Azoospermia,Behcet's Disease, Behcet's syndrome, Berger's Disease, BullousPemphigoid, Cardiomyopathy, Cardiovascular disease, Celiac Sprue/Coeliacdisease, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronicidiopathic polyneuritis, Chronic Inflammatory Demyelinating,Polyradicalneuropathy (CIPD), Chronic relapsing polyneuropathy(Guillain-Barré syndrome), Churg-Strauss Syndrome (CSS), CicatricialPemphigoid, Cold Agglutinin Disease (CAD), COPD, CREST syndrome, Crohn'sDisease, Dermatitis, Herpetiformus, Dermatomyositis, diabetes, DiscoidLupus, Eczema, Epidermolysis bullosa acquisita, Essential MixedCryoglobulinemia, Evan's Syndrome, Exopthalmos, Fibromyalgia,Goodpasture's Syndrome, graft-related disease or disorder, Graves'Disease, GVHD, Hashimoto's Thyroiditis, Idiopathic Pulmonary Fibrosis,Idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy,immunoproliferative disease or disorder (e.g., psoriasis), Inflammatorybowel disease (IBD), Insulin Dependent Diabetes Mellitus (IDDM),Interstitial lung disease, juvenile diabetes, Juvenile Arthritis,juvenile idiopathic arthritis (JIA), Kawasaki's Disease, Lambert-EatonMyasthenic Syndrome, Lichen Planus, lupus, Lupus Nephritis, LymphoscyticLypophisitis, Ménière's Disease, Miller Fish Syndrome/acute disseminatedencephalomyeloradiculopathy, Mixed Connective Tissue Disease, MultipleSclerosis (MS), muscular rheumatism, Myalgic encephalomyelitis (ME),Myasthenia Gravis, Ocular Inflammation, Pemphigus Foliaceus, PemphigusVulgaris, Pernicious Anaemia, Polyarteritis Nodosa, Polychondritis,Polyglandular Syndromes (Whitaker's syndrome), Polymyalgia Rheumatica,Polymyositis, Primary Agammaglobulinemia, Primary BiliaryCirrhosis/Autoimmune cholangiopathy, Psoriasis, Psoriatic arthritis,Raynaud's Phenomenon, Reiter's Syndrome/Reactive arthritis, Restenosis,Rheumatic Fever, rheumatic disease, Rheumatoid Arthritis, Sarcoidosis,Schmidt's syndrome, Scleroderma, Sjörgen's Syndrome, Solid-organtransplant rejection (kidney, heart, liver, lung, etc.), Stiff-ManSyndrome, Systemic Lupus Erythematosus (SLE), systemic scleroderma,Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis,Thyroiditis, Type 1 diabetes, Type 2 diabetes, Ulcerative colitis,Uveitis, Vasculitis, Vitiligo, Wegener's Granulomatosis, and preventingor suppressing an immune response associated with rejection of a donortissue, cell, graft, or organ transplant by a recipient subject.

The invention also provides for the use of a polypeptide, conjugate,nucleic acid, vector, or cell of the invention for the manufacture of amedicament for inhibiting interaction of CD80-positive cells and/orCD86-positive cells with CD28-positive and/or CTLA-4-positive T cells.In another aspect, the invention includes the use of a polypeptide,conjugate, nucleic acid, vector, or cell of the invention for themanufacture of a medicament for the treatment of a tissue or organtransplant rejection (e.g., solid organ transplant rejection (e.g.,kidney, lung, liver, heart, etc.)) in a mammal.

Assessing Immune Responses

Immune responses suppressed by a polypeptide, nucleic acid, vector,virus, pseudovirus, VLP, or composition of the invention can be measuredby any suitable technique. Examples of useful techniques in assessinghumoral immune responses include flow cytometry, immunoblotting assays,immunohistochemistry assays, immunoprecipitation assays,radioimmunoassays (RIA), and enzyme immunoassays. Enzyme immunoassaysinclude enzyme-linked immunoflow assays (ELIFA) and enzyme-linkedimmunosorbent assays (ELISA), including sandwich ELISA and competitiveELISA assays. HPLC and capillary electrophoresis (CE) also can beutilized in immunoassays to detect complexes of antibodies and targetsubstances. General guidance performing such techniques and relatedprinciples are described in, e.g., Harlow and Lane (1988) ANTIBODIES, ALABORATORY MANUAL, Cold Spring Harbor Publications, New York, Hampton Ret al. (1990) SEROLOGICAL METHODS A LABORATORY MANUAL, APS Press, St.Paul Minn., Stevens (1995) CLINICAL IMMUNOLOGY AND SEROLOGY: ALABORATORY PERSPECTIVE, CRC press, Bjerrum (1988) HANDBOOK OFIMMUNOBLOTTING OF PROTEINS, Vol. 2, Zoa (1995) DIAGNOSTICIMMUNOPATHOLOGY: LABORATORY PRACTICE AND CLINICAL APPLICATION, CambridgeUniversity Press, Folds (1998) CLINICAL DIAGNOSTIC IMMUNOLOGY: PROTOCOLSIN QUALITY ASSURANCE AND STANDARDIZATION Blackwell Science Inc., Bryant(1992) LABORATORY IMMUNOLOGY & SEROLOGY 3rd edition, W B Saunders Co.,and Maddox D E et al. (1983) J. Exp. Med. 158:1211. Guidance withrespect to ELISA techniques and related principles are described in,e.g., Reen (1994) Methods Mol. Biol. 32:461-6, Goldberg et al. (1993)Curr. Opin. Immunol. 5(2):278-81, Voller et al. (1982) Lab. Res. MethodsBiol. Med. 5:59-81, Yolken et al. (1983) Ann. NY Acad. Sci. 420:381-90,Vaughn et al. (1999) Am. J. Trop. Med. Hyg. 60(4):693-8, and Kuno et al.(1991) J. Virol. Methods 33(1-2):101-13. Guidance with respect to flowcytometry techniques is provided in, e.g., Diamond (2000) IN LIVINGCOLOR: PROTOCOLS IN FLOW CYTOMETRY AND CELL SORTING, Springer Verlag,Jaroszeki (1998) FLOW CYTOMETRY PROTOCOLS, 1st Ed., Shapiro (1995)PRACTICAL FLOW CYTOMETRY, 3rd edition, Rieseberg et al. (2001) Appl.Microbiol. Biotechnol. 56(3-4):350-60, Scheffold and Kern (2000) J.Clin. Immunol. 20(6):400-7, and McSharry (1994) Clin. Microbiol. Rev.(4):576-604.

Cytotoxic and other T cell immune responses also can be measured by anysuitable technique. Examples of such techniques include ELISpot assay(particularly, IFN-gamma ELISpot), intracellular cytokine staining (ICC)(particularly in combination with FACS analysis), CD8+ T cell tetramerstaining/FACS, standard and modified T cell proliferation assays,chromium release CTL assay, limiting dilution analysis (LDA), and CTLkilling assays. Guidance and principles related to T cell proliferationassays are described in, e.g., Plebanski and Burtles (1994) J. Immunol.Meth. 170:15, Sprent et al. (2000) Philos. Trans. R. Soc. Lond. B Biol.Sci. 355(1395):317-22 and Messele et al. (2000) Clin. Diagn. Lab.Immunol. 7(4):687-92. LDA is described in, e.g., Sharrock et al. (1990)Immunol. Today 11:281-286. ELISpot assays and related principles aredescribed in, e.g., Czerinsky et al. (1988) J. Immunol. Meth. 110:29-36,Olsson et al. (1990) J. Clin. Invest. 86:981-985, Schmittel et al.(2001) J. Immunol. Meth. 247(1-2):17-24, Ogg and McMichael (1999)Immunol. Lett. 66(1-3):77-80, Schmittel et al. (2001) J. Immunol. Meth.247(1-2):17-24, Kurane et al. (1989) J. Exp. Med. 170(3):763-75, Chainet al. (1987) J. Immunol. Meth. 99(2):221-8, Czerkinsky et al. (1988) J.Immunol. Meth. 110:29-36, and U.S. Pat. Nos. 5,750,356 and 6,218,132.Tetramer assays are discussed in, e.g., Skinner et al. (2000) J.Immunol. 165(2):613-7. Other T cell analytical techniques are describedin Hartel et al. (1999) Scand. J. Immunol. 49(6):649-54 and Parish etal. (1983) J. Immunol. Meth. 58(1-2):225-37.

T cell activation also can be analyzed by measuring CTL activity orexpression of activation antigens such as IL-2 receptor, CD69 or HLA-DRmolecules. Proliferation of purified T cells can be measured in a mixedlymphocyte reaction (MLR) assay; such assays are well-known in the art.

ELISpot assays measure the number of T-cells secreting a specificcytokine, such as IFN-γ or TNF-α, which serves as a marker of T-celleffectors. Cytokine-specific ELISA kits are commercially available(e.g., an IFN-γ-specific ELISPot is available through R&D Systems,Minneapolis, Minn.).

Additional methods for assessing and measuring the ability of moleculesof the invention (e.g., polypeptides of the invention, including, e.g.,soluble mutant CTLA-4-Ig fusion proteins of the invention) to suppressor inhibit T cell activation and/or T cell proliferation are describedin Examples 5-8 in the Examples section below.

Methods of Administration

In any of the methods described herein, an injectable pharmaceuticalcomposition comprising a suitable pharmaceutically acceptable excipientor carrier (e.g., PBS) and an effective amount of a molecule of theinvention, such as a polypeptide (e.g., mutant CTLA-4 ECD or monomeric,dimeric, or multimeric mutant CTLA-4-Ig) or conjugate of the invention,can be administered parenterally, intramuscularly, intraperitoneally,intravenously, subdermally, transdermally, subcutaneously, orintradermally to a host. Alternatively, biolistic protein deliverytechniques (vaccine gun delivery) can be used (examples of which arediscussed elsewhere herein). Any other suitable technique also can beused. Polypeptide administration can be facilitated via liposomes. Anysuch delivery technique can be used to deliver a polypeptide orconjugate of the invention in conjunction with any therapeutic orprophylactic method described herein.

While the following discussion is primarily directed to nucleic acids,it will be understood that it applies equally to nucleic acid vectors ofthe invention. A nucleic acid of the invention or composition thereofcan be administered to a host by any suitable administration route. Insome aspects of the invention, administration of the nucleic acid isparenteral (e.g., subcutaneous (s.c.), intramuscular (i.m.), orintradermal (i.d.)), topical, or transdermal. The nucleic acid can beintroduced directly into a tissue, such as muscle, by injection using aneedle or other similar device. See, e.g., Nabel et al. (1990), supra;Wolff et al. (1990) Science 247:1465-1468), Robbins (1996) Gene TherapyProtocols, Humana Press, NJ, and Joyner (1993) Gene Targeting: APractical Approach, IRL Press, Oxford, England, and U.S. Pat. Nos.5,580,859 and 5,589,466. Other methods such as “biolistic” orparticle-mediated transformation (see, e.g., U.S. Pat. Nos. 4,945,050and 5,036,006, Sanford et al., J. Particulate Sci. Tech. 5:27-37 (1987),Yang et al., Proc. Natl. Acad. Sci. USA 87:9568-72 (1990), and Williamset al., Proc. Natl. Acad. Sci. USA 88:2726-30 (1991)). These methods areuseful not only for in vivo introduction of DNA into a subject, such asa mammal, but also for ex vivo modification of cells for reintroductioninto a mammal (which is discussed further elsewhere herein).

For standard gene gun administration, the vector or nucleic acid ofinterest is precipitated onto the surface of microscopic metal beads.The microprojectiles are accelerated with a shock wave or expandinghelium gas, and penetrate tissues to a depth of several cell layers. Forexample, the Accel™ Gene Delivery Device manufactured by Agacetus, Inc.Middleton Wis. is suitable for use in this embodiment. The nucleic acidor vector can be administered by such techniques, e.g., intramuscularly,intradermally, subdermally, subcutaneously, and/or intraperitoneally.Additional devices and techniques related to biolistic delivery Int'lPatent Appn. Publ. Nos. WO 99/2796, WO 99/08689, WO 99/04009, and WO98/10750, and U.S. Pat. Nos. 5,525,510, 5,630,796, 5,865,796, and6,010,478.

The nucleic acid can be administered in association with atransfection-facilitating agent, examples of which were discussed above.The nucleic acid can be administered topically and/or by liquid particledelivery (in contrast to solid particle biolistic delivery). Examples ofsuch nucleic acid delivery techniques, compositions, and additionalconstructs that can be suitable as delivery vehicles for the nucleicacids of the invention are provided in, e.g., U.S. Pat. Nos. 5,591,601,5,593,972, 5,679,647, 5,697,901, 5,698,436, 5,739,118, 5,770,580,5,792,751, 5,804,566, 5,811,406, 5,817,637, 5,830,876, 5,830,877,5,846,949, 5,849,719, 5,880,103, 5,922,687, 5,981,505, 6,087,341,6,107,095, 6,110,898, and Int'l Pat. Appn. Publ. Nos. WO 98/06863, WO98/55495, and WO 99/57275.

Alternatively, the nucleic acid can be administered to the host by wayof liposome-based gene delivery. Exemplary techniques and principlesrelated to liposome-based gene delivery is provided in, e.g., Debs andZhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques6(7):682-691; Rose U.S. Pat. No. 5,279,833; Brigham (1991) WO 91/06309;Brigham et al. (1989) Am. J. Med. Sci. 298:278-281; Nabel et al. (1990)Science 249:1285-1288; Hazinski et al. (1991) Am. J. Resp. Cell Molec.Biol. 4:206-209; and Wang and Huang (1987) Proc. Natl. Acad. Sci. USA84:7851-7855), and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA84:7413-7414). Suitable liposome pharmaceutically acceptablecompositions that can be used to deliver the nucleic acid are furtherdescribed elsewhere herein.

Any amount of nucleic acid of the invention can be used in the methodsof the invention. For example, sufficient nucleic acid may be formulatedin a pharmaceutically acceptable excipient or carrier and administeredto a subject such that the encoded polypeptide or conjugate is producedin the subject in an amount believed effective to, for example, suppressimmune response in the subject, inhibit interaction between endogenousB7-positive cells and CD28-positive cells in the subject, or inhibitrejection of a tissue, cell, organ, or graft transplant. In one format,where the nucleic acid is administered by injection, about 50 micrograms(μg) to 100 mg nucleic acid is administered. In one exemplaryapplication, to suppress an immune response, a pharmaceuticalcomposition comprising PBS and an amount of a DNA vector that encodes aneffective amount of a mutant CTLA-4 polypeptide is administered byinjection or electroporation or other suitable delivery method (e.g.,gene gun, impressing through the skin, and lipofection) to a subject inneed of treatment (e.g., a subject suffering from an immune systemdisease or disorder in which immunosuppressive treatment is desirable).An exemplary vector is shown in FIG. 1.

The amount of DNA plasmid for use in the methods of the invention whereadministration is via a gene gun, e.g., is often from about 100 to about1000 times less than the amount used for direct injection (e.g., viastandard needle injection). Despite such sensitivity, at least about 1μg of the nucleic acid may be used in such biolistic deliverytechniques.

RNA or DNA viral vector systems can be useful for delivery nucleic acidsencoding polypeptides of the invention. Viral vectors can beadministered directly to a subject in vivo or they can be used to treatcells in vitro and the modified cells are administered to the subject inan ex vivo format. Useful viral vectors include those discussed above,such as adeno-associated, adenoviral, retroviral, lentivirus, and herpessimplex virus vectors. With such viral vectors, a nucleic acid of theinvention can be readily transferred into target cells and tissues ofthe subject. Additionally, with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, a nucleic acid of theinvention can be integrated into the host genome may be possible,thereby resulting in continuing expression of the inserted nucleic acid.

Delivery of a viral vector of the invention comprising at least onenucleic acid of the invention to a subject is believed capable ofsuppressing an immune response in the subject to whom the vector isadministered. Optionally, some prophylactic and/or therapeutic methodsof the invention are practiced with a dosage of a suitable viral vectorsufficient to inhibit a detectable immune response. Any suitable viralvector comprising a nucleic acid of the invention, in any suitableconcentration, can be used to suppress the immune response. For example,to the subject host can be administered a population of retroviralvectors (examples of which are described in, e.g., Buchscher et al.(1992) J. Virol. 66(5) 2731-2739, Johann et al. (1992) J. Virol. 66(5):1635-1640 (1992), Sommerfelt et al., (1990) Virol. 176:58-59, Wilsonet al. (1989) J. Virol. 63:2374-2378, Miller et al., J. Virol.65:2220-2224 (1991), Wong-Staal et al., PCT/US94/05700, Rosenburg andFauci (1993) in FUNDAMENTAL IMMUNOLOGY, THIRD EDITION Paul (ed.) RavenPress, Ltd., New York and the references therein), an AAV vector (asdescribed in, e.g., West et al. (1987) Virology 160:38-47, Kotin (1994)Human Gene Therapy 5:793-801, Muzyczka (1994) J. Clin. Invest. 94:1351,Tratschin et al. (1985) Mol. Cell. Biol. 5(11):3251-3260, U.S. Pat. Nos.4,797,368 and 5,173,414, and Int'l Patent Appn Publ. No. WO 93/24641),or an adenoviral vector (as described in, e.g., Berns et al. (1995) Ann.NY Acad. Sci. 772:95-104; Ali et al. (1994) Gene Ther. 1:367-384; andHaddada et al. (1995) Curr. Top. Microbiol. Immunol. 199 (Pt3):297-306), such that immunosuppressive levels of expression of thenucleic acid included in the vector result, thereby resulting in thedesired immunosuppressive response. Other suitable types of viralvectors are described elsewhere herein (including alternative examplesof suitable retroviral, AAV, and adenoviral vectors).

Suitable infection conditions for these and other types of viral vectorparticles are described in, e.g., Bachrach et al., J. Virol., 74(18),8480-6 (2000), Mackay et al., J. Virol., 19(2), 620-36 (1976), andFIELDS VIROLOGY, supra. Additional techniques useful in the productionand application of viral vectors are provided in, e.g., “PracticalMolecular Virology: Viral Vectors for Gene Expression” in METHODS INMOLECULAR BIOLOGY, vol. 8, Collins, M. Ed., (Humana Press 1991), VIRALVECTORS: BASIC SCIENCE AND GENE THERAPY, 1st Ed. (Cid-Arregui et al.,Eds.) (Eaton Publishing 2000), “Viral Expression Vectors,” in CURRENTTOPICS IN MICROBIOLOGY AND IMMUNOLOGY, Oldstone et al., Eds.(Springer-Verlag, NY, 1992), and “Viral Vectors” in CURRENTCOMMUNICATIONS IN BIOTECHNOLOGY, Gluzman and Hughes, eds. (Cold SpringHarbor Laboratory Press, 1988).

The toxicity and therapeutic efficacy of vectors or viruses that includeone or more molecules of the invention are determined using standardpharmaceutical procedures in cell cultures or experimental animals. Onecan determine the MLD₅₀ (the minimum dose lethal to 50% of thepopulation) and/or the ED₅₀ (the dose therapeutically effective in 50%of the population) using procedures presented herein and those otherwiseknown in the art. See also S. Plotkin and W. Orenstein, VACCINES (W. B.Saunders Co. 1999 3d ed.) for suggested doses for known viral vaccines.Nucleic acids, polypeptides, proteins, fusion proteins, transduced cellsand other formulations of the present invention can be administered inan amount determined, e.g., by the MLD₅₀ of the formulation, and theside-effects thereof at various concentrations, as applied to the massand overall health of the patient. Thus, for example, the inventionprovides a method of inducing an immune response by administering a doseequal or greater to the ED₅₀ of a pharmaceutically acceptablecomposition comprising a population of virus-like particles or viruses(e.g., attenuated or replication-deficient virus) that comprises apolypeptide or nucleic acid of the invention. Administration can beaccomplished via single dose or divided doses (either byco-administration, serial administration, or combinations thereof).Administration techniques and protocols are described in, e.g., Plotkin(VACCINES) supra and other references cited herein. In a related sense,techniques for assessing dosage of the nucleic acid, polypeptide,vector, and cell compositions effective for inducing immunity aredescribed in, e.g., European Patent Appn No. 1 156 333 and referencescited therein.

The viral vector can be targeted to particular tissues, cells, and/ororgans of a subject, e.g., mammal. Examples of such vectors aredescribed above. For example, the viral vector or nucleic acid vectorcan be used to selectively deliver the nucleic acid sequence of theinvention to monocytes, dendritic cells, cells associated with dendriticcells (e.g., keratinocytes associated with Langerhans cells), T-cells,and/or B-cells. The viral vector can be a replication-deficient viralvector. The viral vector particle also can be modified to reduce hostimmune response to the viral vector, thereby achieving persistent geneexpression. Such “stealth” vectors are described in, e.g., Martin, Exp.Mol. Pathol. 66(1):3-7 (1999), Croyle et al., J. Virol. 75(10):4792-801(2001), Rollins et al., Hum. Gene Ther. 7(5):619-26 (1996), Ikeda etal., J. Virol. 74(10):4765-75 (2000), Halbert et al., J. Virol.74(3):1524-32 (2000), and Int'l Patent Appn Publ. No. WO 98/40509.Alternatively or additionally, the viral vector particles can beadministered by a strategy selected to reduce host immune response tothe vector particles. Strategies for reducing immune response to theviral vector particle upon administration to a host are provided in,e.g., Maione et al., Proc. Natl. Acad. Sci. USA 98(11):5986-91 (2001),Morral et al., Proc. Natl. Acad. Sci. USA 96(22):2816-21 (1999), Pastoreet al., Hum. Gene Ther. 10(11):1773-81 (1999), Morsy et al., Proc. Natl.Acad. Sci. USA 95(14):7866-71 (1998), Joos et al., Hum. Gene Ther.7(13):1555-66 (1996), Kass-Eisler et al., Gene Ther. 3(2):154-62 (1996),U.S. Pat. Nos. 6,093,699, 6,211,160, 6,225,113, U.S. Patent ApplicationPubl. No. 2001-0066947A1.

The skin and muscle are generally preferred targets for administrationof the polypeptides, conjugates, nucleic acids, and vectors of theinvention, by any suitable technique. Thus, the delivery of apolypeptide, conjugate, nucleic acid, or vector of the invention into orthrough the skin of a subject (e.g., mammal), is a feature of theinvention. Such molecules of the invention can be administered in apharmaceutically acceptable injectable solution into or through theskin, e.g., intramuscularly, or intraperitoneally. Administration canalso be accomplished by transdermal devices, or, more typically,biolistic delivery of the polypeptide, conjugate, nucleic acid, and/orvector to, into, or through the skin of the subject or into exposedmuscle of the subject. Transdermal devices provided by the invention,described elsewhere herein, for example, can be applied to the skin of ahost for a suitable period such that sufficient transfer of apolynucleotide and/or vector to the subject occurs, thereby suppressingan immune response in the subject or inhibiting rejection of a graft,cell, or tissue transplant. Muscular administration is more typicallyfacilitated by injection of a liquid solution comprising a polypeptide,polynucleotide, or vector of the invention. Particular cells that can betargeted include dendritic cells, other APCs, B cells, monocytes, Tcells (including T helper cells), and cells associated with such immunesystem cells (e.g., keratinocytes or other skin cells associated withLangerhans cells). Targeting of vectors and nucleic acids of theinvention is described elsewhere herein. Such targeted administrationcan be performed with nucleic acids or vectors comprising nucleic acidsoperably linked to cell and/or tissue-specific promoters, examples ofwhich are known in the art.

The polynucleotide of the invention can be administered by any suitabledelivery system, such that expression of a recombinant polypeptideoccurs in the host resulting in an suppression of an immune response,inhibition of interaction between B7-positive cells and CD28-positive,or inhibition of tissue, cell, organ, or graft transplant rejection. Forexample, an effective amount of a population of bacterial cellscomprising a nucleic acid of the invention can be administered to asubject, resulting in expression of a recombinant mutant CTLA-4polypeptide of the invention, and suppression of an immune response inthe subject. Bacterial cells developed for mammalian gene delivery areknown in the art.

Administration of a polynucleotide or vector of the invention to asubject is facilitated by application of electroporation to an effectivenumber of cells or an effective tissue target, such that the nucleicacid and/or vector is taken up by the cells, and expressed therein,resulting in production of a recombinant polypeptide of the inventiontherein and subsequent suppression of an immune response in the subject.

Production and Purification Methods

The invention further provides methods of making and purifying thepolypeptides, nucleic acids, vectors, and cells of the invention. In oneaspect, the invention provides a method of making a recombinantpolypeptide of the invention by introducing a nucleic acid of theinvention into a population of cells in a culture medium, culturing thecells in the medium (for a time and under conditions suitable fordesired level of gene expression) to produce the polypeptide, andisolating the polypeptide from the cells, culture medium, or both. Thenucleic acid is typically operatively linked to a regulatory sequenceeffective to express the polypeptide encoded by the nucleic acid.

The polypeptide can be isolated from cell lysates, cell supernatants,and/or cell culture medium a variety of suitable techniques known in theart, including, e.g., various chromatography of cell lysates and/or cellsupernatants. For example, the polypeptide can be isolated from celllysates and/or cell culture medium by first concentrating the culturemedium using centrifugal filters (Amicon), alternatively, byprecipitating the polypeptides with ammonium sulfate or polyethyleneglycol and then resuspending the polypeptides in PBS or other suitablebuffers. The polypeptide can then be purified using eithersize-exclusion chromatography on Sephacryl S-400 column (AmershamBiosciences) as described in, e.g., Hjorth, R. and J. Moreno-Lopez, J.Virol. Methods 5:151-158 (1982), or another affinity chromatography, orby centrifugation through 20-60% sucrose gradients as described in,e.g., Konish et al., Virology 188:714-720 (1992). Fractions containingthe desired polypeptides can be identified by ELISA or SDS-PAGE followedby protein silver stain and immunoblotting. The desired fractions arepooled and further concentrated. Sucrose in gradient centrifugationfractions can be removed using PD-10 column (Amersham Biosciences) gelfiltration. Additional purification techniques include those describedin the Examples below and hydrophobic interaction chromatography (Diogo,M. M, et al., J. Gene Med. 3:577-584 (2001)), and any other suitabletechnique known in the art.

Any suitable purification technique that is known in the art can also beused. Polypeptide purification methods known in the art include thoseset forth in, e.g., Sandana (1997) BIOSEPARATION OF PROTEINS, AcademicPress, Inc., Bollag et al. (1996) PROTEIN METHODS, 2 Edition Wiley-Liss,NY, Walker (1996) THE PROTEIN PROTOCOLS HANDBOOK Humana Press, NJ,Harris and Angal (1990) PROTEIN PURIFICATION APPLICATIONS: A PRACTICALAPPROACH IRL Press at Oxford, Oxford, England, Scopes (1993) PROTEINPURIFICATION: PRINCIPLES AND PRACTICE 3 Edition Springer Verlag, NY,Janson and Ryden (1998) PROTEIN PURIFICATION: PRINCIPLES, HIGHRESOLUTION METHODS AND APPLICATIONS, Second Edition Wiley-VCH, NY; andWalker (1998) PROTEIN PROTOCOLS ON CD-ROM Humana Press, NJ. Cellssuitable for polypeptide production are known in the art and arediscussed elsewhere herein (e.g., Vero cells, 293 cells, BHK, CHO (e.g.,CHO-K1), and COS cells can be suitable). Cells can be lysed by anysuitable technique including, e.g., sonication, microfluidization,physical shear, French press lysis, or detergent-based lysis.

In one aspect, the invention provides a method of purifying apolypeptide of the invention, which comprises transforming a suitablehost cell with a nucleic acid of the invention (e.g., a recombinantnucleic acid that encodes a recombinant polypeptide comprising thepolypeptide sequence of SEQ ID NO:1) in the host cell (e.g., a CHO cellor 293 cell), lysing the cell by a suitable lysis technique (e.g.,sonication, detergent lysis, or other appropriate technique), andsubjecting the lysate to affinity purification with a chromatographycolumn comprising a resin that includes at least one novel antibody ofthe invention (usually a monoclonal antibody of the invention) orantigen-binding fragment thereof, such that the lysate is enriched forthe desired polypeptide (e.g., a polypeptide comprising the polypeptidesequence of SEQ ID NO:1).

In another aspect, the invention provides a method of purifying suchtarget polypeptides, which method differs from the above-describedmethod in that a nucleic acid comprising a nucleotide sequence encodinga fusion protein that comprises a polypeptide of the invention (e.g.,SEQ ID NO:1) and a suitable tag (e.g., an e-epitope/his tag), andpurifying the polypeptide by immunoaffinity, lentil-lectin affinitycolumn chromatography, immobilized metal affinity chromatography (IMAC),or metal-chelating affinity chromatography (MCAC) enrichment techniques.Additional purification methods are disclosed elsewhere herein.

In another aspect, the invention provides a method of producing apolypeptide of the invention, which method comprises introducing into apopulation of cells a recombinant expression vector comprising a nucleicacid of the invention, culturing the cells in a culture medium underappropriately sufficient conditions for expression of the nucleic acidfrom the vector and production of the polypeptide encoded by the nucleicacid, and isolating the polypeptide from the cells, culture medium, orboth. The cells chosen are based on the desired processing of thepolypeptide and based on the appropriate vector (e.g., E. coli cells arepreferred for bacterial plasmids, whereas 293 cells are preferred formammalian shuttle plasmids and/or adenoviruses, particularlyE1-deficient adenoviruses).

In yet another aspect, the invention includes a method of producing apolypeptide, the method comprising: (a) introducing into a population ofcells a recombinant expression vector comprising at least one nucleicacid of the invention the encodes a polypeptide of the invention; (b)administering the expression vector into a mammal; and (c) isolating thepolypeptide from the mammal or from a byproduct of the mammal.

A polypeptide of the invention can also be produced by culturing a cellor population of cells of the invention (which, e.g., have beentransformed with a nucleic acid of the invention that encodes suchpolypeptide) under conditions sufficient for expression of thepolypeptide and recovering the polypeptide expressed in or by the cellusing standard techniques known in the art.

In another aspect, the invention provides a method of producing apolypeptide of the invention, which comprises (a) introducing into apopulation of cells a nucleic acid of the invention, wherein the nucleicacid is operatively linked to a regulatory sequence effective to producethe polypeptide encoded by the nucleic acid; (b) culturing the cells ina culture medium to produce the polypeptide; and (c) isolating thepolypeptide from the cells or culture medium. Also included is acultured cell into which has been introduced a vector of the invention(e.g., an expression vector of the invention).

Also included is a method of producing a polypeptide of the inventionwhich comprises introducing a nucleic acid encoding said polypeptideinto a population of cells in a medium, which cells are permissive forexpression of the nucleic acid, maintaining the cells under conditionsin which the nucleic acid is expressed, and thereafter isolating thepolypeptide from the medium.

In another aspect, the invention provides a method of making a fusionprotein. The method comprises: (1) culturing a host cell transformedwith a nucleic acid in a culture medium, wherein the nucleic acidcomprises: (i) a first nucleotide sequence that encodes a polypeptidehaving at least 95% identity to a polypeptide sequence selected from thegroup consisting of SEQ ID NOS:1-73, which polypeptide binds CD86 and/orCD80, and/or an extracellular domain of either CD86 or CD80, and (ii) asecond nucleotide sequence encoding an Ig Fc polypeptide comprising ahinge region, CH2 domain, and CH3 domain, whereby the nucleic acid isexpressed and a fusion protein is produced; and (2) recovering thefusion protein. Any Ig Fc polypeptide may be any employed, includinge.g., an IgG1 Fc, IgG2 Fc, IgG4 Fc, or mutant Ig Fc polypeptide. In somesuch methods, the nucleic acid further comprises a third nucleotidesequence that encodes a secretory or signal peptide operably linked tothe fusion protein, and the fusion protein is secreted from the hostcell as a disulfide-bonded fusion protein dimer comprising identicalfirst and second fusion proteins, and the disulfide-bonded fusionprotein dimer is recovered from the culture medium. In some suchmethods, the disulfide-bonded fusion protein dimer is formed via acovalent disulfide bond between a cysteine residue of the first fusionprotein and a cysteine residue of the second fusion protein. In somesuch methods, the fusion protein is recovered from the culture medium,host cell, or host cell periplasm.

In another aspect, the invention provides an isolated or recombinantnucleic acid molecule comprising a nucleotide sequence which encodes (i)a first polypeptide comprising a polypeptide sequence having at least95% sequence identity to at least one polypeptide sequence selected fromthe group consisting of SEQ ID NOS:1-73, wherein the first polypeptidebinds CD80 and/or CD86 and/or an extracellular domain of either or both,and (ii) a second polypeptide comprising a comprising a hinge region,CH2 domain, and CH3 domain of an IgG polypeptide. The second polypeptidemay comprise any suitable Ig polypeptide discussed elsewhere herein,including, e.g., that comprising the polypeptide sequence of SEQ IDNO:184 or SEQ ID NO:218.

In another aspect, the invention provides a method of making a solublefusion protein dimer. The method comprises culturing a host celltransformed with an expression vector comprising a nucleotide sequencethat encodes a soluble fusion protein dimer of the invention. Exemplaryfusion proteins include those comprising the polypeptide sequence of anyof SEQ ID NOS:74-79, 197-200, 205-214, and 219-222. The vector includesa nucleotide sequence that facilitates expression of the fusion protein(e.g., a nucleotide sequence encoding a signal peptide). The fusionprotein is secreted from the host cell as a disulfide-bonded fusionprotein dimer comprising two identical fusion proteins, and thedisulfide-bonded fusion protein dimer is recovered from the culturemedium. In some such methods, the disulfide-bonded fusion protein dimeris formed via a covalent disulfide bond between a cysteine residue oneach fusion protein. The fusion protein dimer is typically recoveredfrom the culture medium, host cell, or host cell periplasm. Example 12provides an exemplary procedure for creating a stably transfected cellline expressing a mutant CTLA4-Ig fusion protein of the invention,producing the mutant CTLA4-Ig fusion protein, and the purifying themutant fusion protein from culture.

In addition to recombinant production, the polypeptides of the inventionmay be produced by direct peptide synthesis using solid-phase techniques(see, e.g., Stewart et al. (1969) SOLID-PHASE PEPTIDE SYNTHESIS, W.H.Freeman Co, San Francisco and Merrifield J. (1963) J. Am. Chem. Soc.85:2149-2154). Peptide synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer, Foster City, Calif.) in accordance with the instructions providedby the manufacturer. For example, subsequences may be chemicallysynthesized separately and combined using chemical methods to produce apolypeptide of the invention or fragments thereof. Alternatively,synthesized polypeptides may be ordered from any number of companiesthat specialize in production of polypeptides. Most commonly,polypeptides of the invention are produced by expressing coding nucleicacids and recovering polypeptides, e.g., as described above.

The invention includes a method of producing a polypeptide of theinvention comprising introducing a nucleic acid of the invention, avector of the invention, or a combination thereof, into an animal, suchas a mammal (including, e.g., rat, nonhuman primate, bat, marmoset, pig,or chicken), such that a polypeptide of the invention is expressed inthe animal, and the polypeptide is isolated from the animal or from abyproduct of the animal. Isolation of the polypeptide from the animal oranimal byproduct can be by any suitable technique, depending on theanimal and desired recovery strategy. For example, the polypeptide canbe recovered from sera of mice, monkeys, or pigs expressing thepolypeptide of the invention. Transgenic animals (including theaforementioned mammals) comprising at least one nucleic acid of theinvention are provided by the invention. The transgenic animal can havethe nucleic acid integrated into its host genome (e.g., by an AAVvector, lentiviral vector, biolistic techniques performed withintegration-promoting sequences, etc.) or can have the nucleic acid inmaintained epichromosomally (e.g., in a non-integrating plasmid vectoror by insertion in a non-integrating viral vector). Epichromosomalvectors can be engineered for more transient gene expression thanintegrating vectors. RNA-based vectors offer particular advantages inthis respect.

Compositions

The invention further provides novel and useful compositions comprisingat least one component of the invention, such as, e.g., at least onepolypeptide (including, e.g., fusion proteins and multimericpolypeptides), conjugate, nucleic acid, vector, virus, virus-likeparticle (VLP), and/or cell of the invention, or any combination thereofand a carrier, excipient, or diluent. The carrier, excipient or diluentmay be a pharmaceutically acceptable carrier, excipient, or diluent.Such a composition can comprise any suitable amount of any suitablenumber of polypeptides, conjugates, nucleic acids, vectors, viruses,VLPs, and/or cells of the invention. Also provided are pharmaceuticalcompositions comprising at least one polypeptide, conjugate, nucleicacid, vector, virus, VLP, and/or cell, or any combination thereof, and apharmaceutically acceptable carrier, excipient, or diluent. Suchcompositions are useful in the methods of the invention describedherein, including, e.g., methods of suppressing immune responses.

For example, in one non-limiting embodiment, the invention provides acomposition comprising an excipient, diluent, or carrier and at leastone such polypeptide of the invention (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more polypeptides), such as a mutant CTLA-4 ECD polypeptide (e.g.,any of SEQ ID NOS:1-73) or mutant CTLA-4-Ig fusion protein (e.g., any ofSEQ ID NOS:74-79, 197-200, 205-214, and 219-222), wherein the at leastone polypeptide is present in the composition in an amount effective tosuppress an immune response, including, e.g., an immune response(s)involved in transplant rejection and/or autoimmunity, inhibit rejectionof a donated tissue, cell, or organ transplant, or inhibit interactionof endogenous B7-positive cells with CD28-positive T cells in a subjectto whom the composition is administered.

Also included is a pharmaceutical composition comprising apharmaceutically acceptable excipient, diluent, or carrier and aneffective amount of one or more such components of the invention. Theeffective amount may be a therapeutically or prophylactically effectiveamount or dose for use in a therapeutic or prophylactic method describedelsewhere herein, such as a method of treating an autoimmune disease ora method of inhibiting rejection of a tissue, cell, graft, or organtransplant from a donor by a recipient subject.

The composition (or pharmaceutical composition) can be any non-toxiccomposition that does not interfere with the immunosuppressiveproperties of the polypeptide, conjugate, nucleic acid, vector, virus,VLP, or cell of the invention included therein. The composition cancomprise one or more excipients, diluents, or carriers, and thepharmaceutical composition comprises one or more pharmaceuticallyacceptable excipients, diluents, or carriers. A wide variety ofacceptable carriers, diluents, and excipients are known in the art andcan be included in the compositions and pharmaceutical compositions ofthe invention. For example, a variety of aqueous carriers can be used,e.g., distilled or purified water, sterile saline, buffered saline, suchas phosphate-buffered saline (PBS), and the like are advantageous ininjectable formulations of the polypeptide, fusion proteins, conjugate,nucleic acid, vector, virus, VLP, and/or cell of the invention. Numeroussuitable excipients, carriers, and diluents for administration oftherapeutic proteins are known in the art. Such solutions are preferablysterile and generally free of undesirable matter. Compositions may besterilized by conventional, well-known sterilization techniques.Compositions of the invention may comprise pharmaceutically acceptableauxiliary substances, as required, to approximate physiologicalconditions. Such substances include, e.g., pH adjusting agents,buffering agents, and tonicity adjusting agents, including, e.g., sodiumacetate, sodium ascorbate, sodium chloride, potassium chloride, calciumchloride, sodium lactate, and the like. Compositions of the invention,including pharmaceutical compositions, can also include one or morecomponents, such as diluents, fillers, salts, buffers, surfactants,emulsifiers, detergents (e.g., a nonionic detergent or emulsifier, suchas Tween®-20, Tween®-40, Tween®-60, Tween®-80, pluronic F-68, and thelike), stabilizers (e.g., sugars or protein-free amino acids),preservants, tissue fixatives, solubilizers, and/or other materials,suitable for inclusion in a pharmaceutically composition.

Examples of suitable components that may be used in the pharmaceuticalcomposition are described in, e.g., Berge et al., J. Pharm. Sci.66(1):1-19 (1977), Wang and Hanson, J. Parenteral. Sci. Tech. 42:S4-S6(1988), U.S. Pat. Nos. 6,165,779 and 6,225,289, and elsewhere herein.Pharmaceutical compositions also can include preservatives (such asbenzyl alcohol, sodium azide, m-cresol, etc.), antioxidants, metalchelators (such as methionine, EDTA, etc.), and/or other additives knownto those of skill in the art. Examples of suitable pharmaceuticallyacceptable carriers for use in the pharmaceutical compositions aredescribed in, e.g., Urquhart et al., Lancet 16:367 (1980), Lieberman etal., PHARMACEUTICAL DOSAGE FORMS—DISPERSE SYSTEMS (2nd ed., Vol. 3,1998), Ansel et al., PHARMACEUTICAL DOSAGE FORMS & DRUG DELIVERY SYSTEMS(7th ed. 2000), Martindale, THE EXTRA PHARMACOPEIA (31st edition),Remington's PHARMACEUTICAL SCIENCES (16th-20th editions), THEPHARMACOLOGICAL BASIS OF THERAPEUTICS, Goodman and Gilman, Eds. (9thed.—1996), WILSON AND GISVOLDS TEXTBOOK OF ORGANIC MEDICINAL ANDPHARMACEUTICAL CHEMISTRY, Delgado and Remers, Eds. (10th ed.—1998), andU.S. Pat. Nos. 5,708,025 and 5,994,106. Principles of formulatingpharmaceutically acceptable compositions are described in, e.g., Platt,Clin. Lab Med. 7:289-99 (1987), Aulton, PHARMACEUTICS: THE SCIENCE OFDOSAGE FORM DESIGN, Churchill Livingstone (New York) (1988),EXTEMPORANEOUS ORAL LIQUID DOSAGE PREPARATIONS, CSHP (1998), and “DrugDosage,” J. Kans. Med. Soc. 70(1):30-32 (1969). Additionalpharmaceutically acceptable carriers particularly suitable foradministration of vectors are described in, e.g., Int'l Patent AppnPubl. No. WO 98/32859.

Compositions of the invention, including pharmaceutical compositions,can include one or more aqueous carriers or excipients (including, e.g.,pharmaceutically acceptable carriers or excipients) and one or morecomponents, such as one or more buffers, one or more salts, one or moredetergents or emulsifiers, and/or one or more sugars. The buffer systemis typically one suitable to maintain the pH of the composition within arange which is conducive to the stability of the molecule of theinvention present in the composition (e.g., mutant CTLA-4-Ig). Exemplarybuffers for use in the composition include, but are not limited to,e.g., N-2-hydroxyethylpiperazine-N′-2-aminoethane sulfonic acid (HEPES)buffer, citrate buffer (e.g., disodium citrate-trisodium citratemixture, sodium citrate-citric acid mixture, citric acid-trisodiumcitrate mixture, monosodium citrate-disodium citrate mixture, citricacid-monosodium citrate mixture), sodium phosphate buffer (e.g.,disodium phosphate-trisodium phosphate mixture (Na₂HPO₄/Na₃PO₄), sodiumdibasic phosphate-sodium monobasic phosphate mixture), acetate buffer(e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxidemixture), histidine buffer, Tris buffer, Tris-maleate buffer, succinatebuffer (e.g., succinic acid-sodium hydroxide mixture, succinicacid-monosodium succinate mixture, succinic acid-disodium succinatemixture, monosodium succinate-disodium succinate mixture), maleatebuffer, imidazole buffer, tartrate buffer, fumarate buffer, gluconatebuffer, oxalate buffer, lactate buffer, acetate buffer, and the like, ora combination of any thereof (e.g., cocktail of citrate and acetatebuffers, etc.). The concentration of buffer in the composition can beany that is appropriate for the molecule(s) of the invention (e.g., amutant CTLA-4-Ig) included in the composition solution, such as, but notlimited to, e.g., in the range of from about 1 mM to about 100 mM, about1 mM to about 50 mM, about 5 mM to about 50 mM, or about 5 mM to about25 mM, including, e.g., 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM,40 mM, 50 mM (such as, e.g., 20 mM HEPES buffer, 20 mM disodiumcitrate-trisodium citrate buffer, 20 mM succinate buffer, etc.).

Exemplary salts for use in the composition include, but are not limitedto, e.g., water-soluble salts, including an organic salt or inorganicsalt (e.g., water-soluble inorganic salt), such as sodium chloride,magnesium chloride, sodium bicarbonate, potassium chloride, calciumchloride, and ammonium chloride, and the like, or any pharmaceuticallyacceptable or physiologically compatible salt. Exemplary concentrationsof salt in the composition solution include, but are not limited to,e.g., in the range of from about 1 mM to about 150 mM, about 10 mM toabout 125 mM, or about 75 mM to about 125 mM, including, e.g., 10 mM, 50mM, 75 mM, 100 mM, 125 mM, 150 mM (such as, e.g., 100 mM NaCl).

Exemplary sugars or carbohydrates for use in the composition include,but are not limited to, e.g., sucrose, maltose, trehalose, dextrose,mannose, raffinose, lactose, malto dextrin, dextran, saccharose, etc.,in a concentration range including, but not limited to, e.g., from about0.1% to about 10% by weight sugar, about 1% to about 5% by weight sugar,or about 1% to about 3% by weight sugar, including e.g., 0.1%, 0.5%, 1%,1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% by weight sugar(e.g., 2% by weight sucrose, 2% by weight trehalose, or 2% by weightmannose) based on the composition. Exemplary sugar alcohols for use inthe composition include, but are not limited to, e.g., mannitol,sorbitol, glycol, glycerol, arabitol, erythritol, xylitol, ribitol,lactitol, and the like in a concentration range including, but notlimited to, e.g., from about 0.1% to about 10% by weight sugar alcohol,about 1-5%, about 1-3%, including e.g., 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%,3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% by weight sugar alcohol based on thecomposition.

The osmolality of the compositions of the invention, includingpharmaceutical compositions, is typically similar to the serumosmolality of blood, which ranges from about 250 to about 350milliosmoles per kilogram (mOSm/kg) of water. The concentration of saltin the composition is typically less than 125 mM. The salt and sugarconcentrations may be adjusted or varied such that the osmolality of thecomposition is from about 250-350 mOSm/kg of water.

Exemplary detergents or emulsifiers for use in the composition include,but are not limited to, e.g., polysorbates, such as Tween®-20,Tween®-40, Tween®-60, Tween®-80, or pluronic F-68 in a range including,but not limited to, e.g., from about 0.001% to about 0.2% by weight of adetergent or emulsifier based on the composition, including, e.g.,0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%,0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.075%, and 0.1% by weight ofdetergent or emulsifier (e.g., Tween®-20, Tween®-40, Tween®-60,Tween®-80, or pluronic F-68) based on the composition.

Compositions of the invention, including pharmaceutical compositions,can comprise a polymer, such as a PEG molecule, in a concentrationsufficient to reduce or inhibit undesired association between two ormore molecules of the invention, such as, e.g., two or more mutantCTLA-4-Ig fusion protein dimers of the invention. The composition cancomprise two or more different polymers (e.g., PEGs). The polymer (e.g.,PEG molecule) typically has a molecular weight of from about 200 Da toabout 8000 Da (e.g., about 200, 300, 400, 600, 900, 1000, 1450, 3350,4500, or 8000 Da, available from Dow Chemical). The addition of apolymer (e.g., PEG molecule) to the composition is believed to reducethe formation of undesired aggregates, particularly undesired aggregatesof two or more fusion protein dimers of the invention.

Compositions of the invention, including pharmaceutical compositions,can include a cyclic oligosaccharide, such as a cyclodextrin (e.g.,Captisol® (Cydex)). In one aspect, the composition comprises two or moredifferent cyclic oligosaccharides. The addition of cyclicoligosaccharide(s) to the composition improves the solubility,stability, bioavailability, and/or dosing of active pharmaceuticalingredient(s) (e.g., mutant CTLA-4 molecule).

The pH of a composition of the invention, including a pharmaceuticalcomposition, can range from about pH 3 to about pH 10, from about pH 4to about pH 10, from about pH 5 to about pH 9, from about pH 6 to aboutpH 9, from about pH 5.5 to about pH 8.5, from about pH 6.0 to about pH6.7, from about pH 6.0 to about pH 6.5, from about pH 6.2 to about pH8.7, from about pH 6.5 to about pH 8.5, from about pH 6.5 to about pH7.5, from about pH 6.2 to about pH 7.0, from about pH 6.3 to about pH6.8, from about pH 6.4 to about pH 6.8, from about pH 7.0 to about pH8.0, and about pH 7.0 to about pH 7.4. In one aspect, compositionscomprising a molecule of the invention, such as, e.g., a mutantCTLA-4-IgG2, have a pH of pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, 5.5,pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4,pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3,pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2,pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9.0, pH 9.1,pH 9.2, pH 9.3, pH 9.4, pH 9.5, pH 9.6, pH 9.7, pH 9.8, pH, 9.9, or pH10.0.

In one aspect, the invention provides a composition of the inventioncomprising an excipient or carrier (including, e.g., a pharmaceuticalcomposition comprising a pharmaceutically acceptable excipient orcarrier) and an effective amount of any CTLA-4 polypeptide, multimer,dimer, conjugate, fusion protein, or fusion protein dimer of theinvention described throughout and herein, and further comprising abuffer capable of maintaining the pH of the composition within the rangeof about pH 3 to about pH 10, water, optionally a non-ionic detergent,optionally a salt, and optionally a sugar alcohol, monosaccharide,disaccharide, or polysaccharide. Some such compositions are at aphysiological pH. Some such compositions have a pH of from about 4 toabout 7.5, about 5.0 to about 7.5, or from about 6.4 to about 6.6,including, e.g., about pH 6.5, about pH 7.4, or pH 7.5. Some suchcompositions comprise a buffer in a concentration of from about 1 mM toabout 100 mM, about 1 mM to about 50 mM, about 5 mM to about 35 mM,about 10 mM to about 25 mM, including, e.g., about 20 mM, 25 mM, or 30mM. Some such compositions comprise a buffer selected from the groupconsisting of a HEPES buffer, citrate buffer, succinate buffer, acetatebuffer, citrate buffer, maleate buffer, phosphate buffer, and Trisbuffer. Some such compositions comprise a buffer is selected from thegroup consisting of a HEPES buffer, sodium citrate buffer, and sodiumsuccinate buffer. For some such compositions, the pH is from about 6.0to about 6.7 and the buffer is sodium succinate or sodium citrate. Forsome such compositions, the pH is about 7.0 to about 7.7 and the bufferis HEPES. Some such compositions further comprise a sugar alcohol orsaccharide, wherein the saccharide is a monosaccharide, disaccharide(e.g., sucrose or trehalose), or polysaccharide. Some such compositionscomprise a salt present in a concentration of about 1 mM to about 50 mM,including, e.g., about 20 mM, 25 mM, or 30 mM. Some such compositionscomprise a non-ionic detergent, such as, e.g., a non-ionic detergentselected from the group consisting of from the group consisting ofTween®-80, Tween®-60, Tween®-40, Tween®-20, or pluronic F-68.

In some such compositions (including pharmaceutical compositions)described in the paragraph above, the polypeptide, multimer, dimer,conjugate, fusion protein, or fusion protein dimer is present at aconcentration in the range of about 1 mg/ml (weight/volume or w/v) toabout 200 mg/ml (w/v), about 25 mg/ml (w/v) to about 100 mg/ml (w/v),about 50 mg/ml to about 300 mg/ml, optionally about in a range of about50 mg/ml (w/v) to about 100 mg/ml (w/v). Some such compositions comprisean effective amount of the polypeptide, multimer, dimer, conjugate,fusion protein, or fusion protein dimer of from about 0.1 mg/kg to about15 mg/kg, and the composition is administered to a mammal (e.g., human).Some such compositions comprise an effective amount of the polypeptide,multimer, dimer, conjugate, fusion protein, or fusion protein dimer offrom about 0.5 mg/kg to about 10 mg/kg, and the composition isadministered parenterally. Some such compositions comprise an effectiveamount of the polypeptide, multimer, dimer, conjugate, fusion protein,or fusion protein dimer of from about 0.1 mg/kg to about 5 mg/kg, andoptionally about 0.5 mg/kg, and the composition is administeredsubcutaneously. Some such compositions comprise an effective amount ofthe polypeptide, multimer, dimer, conjugate, fusion protein, or fusionprotein dimer of from about 5 mg/kg to about 15 mg/kg (optionally about10 mg/kg), and the composition is administered intravenously. For somesuch compositions, the polypeptide, multimer, dimer, conjugate, fusionprotein, or fusion protein dimer comprises an amino acid sequence havingat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO:36. For somesuch compositions, the polypeptide, multimer, dimer, conjugate, fusionprotein, or fusion protein dimer comprises an amino acid sequence havingat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO:50. Some suchcompositions are sterile and/or are isotonic to blood. Some suchcompositions are liquid compositions. Some such compositions are in aliquid or a dried form, wherein the dried form is selected from thegroup consisting of a lyophilized form, an air-dried form, and aspray-dried form.

In an exemplary aspect, the invention provides a pharmaceuticalcomposition comprising: (i) a CTLA-4-Ig fusion protein of the inventionhaving a concentration of from about 1 mg/ml to about 300 mg/ml (e.g.,about 1 mg/ml to about 100 mg/ml, about 50 mg/ml or about 100 mg/ml,etc.) (optionally a dimeric fusion protein); (ii) a buffer having abuffering capacity of between about pH 5.0 and about pH 9.0 at aconcentration of about 5 mM to about 50 mM; (iii) a pharmaceuticallyacceptable diluent to bring the composition to a designated volume; (iv)a sugar at a concentration of about 0.5% to about 10% by weight sugarbased on the composition; (v) a salt at a concentration of about 1 mM toabout 200 mM; (vi) optionally a non-ionic detergent (e.g., Tween®-20,Tween®-40, Tween®-60, Tween®-80, or pluronic F-68) at a concentration ofabout 0.01 mg/ml to about 0.5 mg/ml, e.g., about 0.01 mg/ml to about 0.1mg/ml; and (vii) optionally a cyclic oligosaccharide (e.g., cyclodextrin(Captisol®), wherein the pH of the composition is in a range of about pH5.0 to about pH 8.0. Exemplary CTLA-4-Ig fusion protein of the inventioninclude those comprising a polypeptide sequence having at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to atleast one polypeptide sequence selected from the group consisting of SEQID NOS:74-79, 197-200, 205-214, and 219-222 (optionally, e.g., selectedfrom the group consisting of SEQ ID NOS:197, 199, 211, and 213), whereinthe fusion protein binds CD80 and/or CD86 and/or an extracellular domainthereof and/or suppresses an immune response. Such fusion proteins maybe in monomeric or dimeric form.

In another aspect, the invention provides a pharmaceutical compositioncomprising: (i) a conjugate comprising a CTLA-4-Ig fusion protein of theinvention (optionally a dimeric fusion protein) and a non-polypeptidemoiety covalently attached to the fusion protein, said conjugate havinga concentration of from about 1 mg/ml to about 300 mg/ml (e.g., about 1mg/ml to about 100 mg/ml, about 50 mg/ml or about 100 mg/ml, etc.); (ii)a buffer having a buffering capacity of between about pH 5.0 and aboutpH 8.0 at a concentration of about 5 mM to about 50 mM; (iii) apharmaceutically acceptable diluent to bring the composition to adesignated volume; (iv) a sugar at a concentration of about 0.5% toabout 10% by weight sugar based on the composition; (v) a salt at aconcentration of about 1 mM to about 200 mM; and (vi) optionally anon-ionic detergent (e.g., Tween®-20, Tween®-40, Tween®-60, Tween®-80,or pluronic F-68) at a concentration of about 0.01 mg/ml to about 0.5mg/ml, e.g., about 0.01 mg/ml to about 0.1 mg/ml, wherein the pH of thecomposition is in a range of about pH 5.0 to about pH 8.0. The conjugatemay comprise one, two, three, four or more non-polypeptide moieties.Each non-polypeptide moiety may comprise a polymer (e.g., PEG or PAO) ora sugar moiety. In some instances, non-polypeptide moiety is a polymermolecule, such as a PEG molecule. The polymer molecule can have anydesired molecular weight dependent on the desired functional effect(e.g., increased half life, decreased association between fusion proteinmolecules, etc.). In some instances, e.g., the polymer is a PEG having amolecular weight of from about 1 kDa to about 100 kDa Da (e.g., 1, 2,2.5, 3, 5, 8, 10, 12, 20, 25, 30, 40, 60 kDa, etc.). The non-polypeptidemoiety (e.g., sugar moiety or polymer molecule) is covalently attachedto an attachment group of an amino acid residue of the fusion proteinusing standard procedures as described above. Exemplary CTLA-4-Ig fusionproteins include those comprising a polypeptide sequence having at least91%, at least 92%, at least 93%, at least 94%, least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or 100% sequence identity toat least one polypeptide sequence selected from the group consisting ofSEQ ID NOS:74-79, 197-200, 205-214, and 219-222 (optionally, e.g.,selected from the group consisting of SEQ ID NOS:197, 199, 211, and213), wherein the fusion protein binds CD80 and/or CD86 and/or anextracellular domain thereof and/or suppresses an immune response. Suchfusion proteins may be in monomeric or dimeric form.

In another aspect, the invention provides a pharmaceutical compositioncomprising: (a) a polypeptide comprising a polypeptide sequence havingat least about 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to apolypeptide sequence selected from the group of SEQ ID NOS:1-73, suchas, e.g., SEQ ID NOS:36 and 50, wherein said polypeptide is present in aconcentration range of about 1 to about 200 mg/ml (w/v); (b) a bufferhaving a buffering capacity of between about pH 5.0 and about pH 8.0 ata concentration range of about 5 mM to about 50 mM; (c) apharmaceutically acceptable diluent to bring the composition to adesignated volume; (d) a sugar at a concentration of 0.5% to 10% byweight; (e) a salt at a concentration of about 1 mM to about 200 mM; and(f) optionally a detergent, wherein the pH is in a range of about pH 5.0to about pH 8.0. In some such pharmaceutical compositions, (a) thepolypeptide comprises the polypeptide sequence of SEQ ID NO:36 presentin a concentration range of from about 50 mg/ml to about 100 mg/ml; (b)the buffer is HEPES buffer present at a concentration of about 20 mM;(c) the pharmaceutically acceptable diluent is water; (d) the sugar issucrose or trehalose at a concentration of 2% by weight; (e) the salt issodium chloride at a concentration of about 100 mM; and (f) optionally adetergent selected from the group consisting of Tween®-80, Tween®-60,Tween®-40, Tween®-20, or pluronic F-68 at a concentration less than orequal to about 0.1 mg/ml, wherein the pH of the composition is about pH7.4. In another such compositions, (a) the polypeptide comprises thepolypeptide sequence of SEQ ID NO:50 present in a concentration range offrom about 50 mg/ml to about 100 mg/ml; (b) the buffer is sodium citratebuffer present at a concentration of about 20 mM; (c) thepharmaceutically acceptable diluent is water; (d) the sugar is sucroseor trehalose at a concentration of 2% by weight; (e) the salt is sodiumchloride at a concentration of about 100 mM; and (f) optionally adetergent selected from the group consisting of Tween®-80, Tween®-60,Tween®-40, Tween®-20, or pluronic F-68 at a concentration less than orequal to about 0.1 mg/ml, wherein the pH is about pH 6.5.

In another aspect, the invention provides a pharmaceutical compositioncomprising: (a) a polypeptide comprising an amino acid sequence havingat least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the amino acid sequence of SEQ ID NO:36; and (b) HEPES orsodium citrate buffer (e.g., disodium citrate-trisodium citrate mixture,sodium citrate-citric acid mixture, citric acid-trisodium citratemixture, monosodium citrate-disodium citrate mixture, or citricacid-monosodium citrate mixture).

Also provided is a pharmaceutical composition comprising: (a) apolypeptide comprising an amino acid sequence having at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NO:36; and (b) a pharmaceutically acceptable excipient or apharmaceutically acceptable carrier, wherein the composition has a pHfrom about 7 to about 8. Some such pharmaceutical compositions have a pHof about 7.4 or 7.5. Some such pharmaceutical compositions compriseHEPES or sodium citrate buffer.

Also provided is a pharmaceutical composition comprising: (a) apolypeptide comprising an amino acid sequence having at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence of SEQ ID NO:50; and (b) sodium citrate buffer (e.g.,disodium citrate-trisodium citrate mixture, sodium citrate-citric acidmixture, citric acid-trisodium citrate mixture, monosodiumcitrate-disodium citrate mixture, or citric acid-monosodium citratemixture).

Also provided is a pharmaceutical composition comprising: (a) apolypeptide comprising an amino acid sequence having at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NO:50; and (b) a pharmaceutically acceptable excipient or apharmaceutically acceptable carrier, wherein the composition has a pHfrom about 6 to about 7. Some such pharmaceutical compositions have a pHof about 6.5. Some such pharmaceutical compositions comprise sodiumcitrate buffer.

In one exemplary aspect, the invention provides a pharmaceuticalcomposition comprising from about 1 mg/ml to about 300 mg/ml of aCTLA-4-Ig fusion protein of the invention (e.g., D3-54-IgG2) (e.g.,about 1 mg/ml to about 100 mg/ml, e.g., about 50 mg/ml or about 100mg/ml) which is typically expressed as a dimeric fusion protein, in 20mM HEPES buffer in water, 100 mM NaCl, 2% by weight sucrose based on thecomposition, pH 7.4, optionally including a non-ionic detergent (e.g.,Tween®-20, Tween®-40, Tween®-60, Tween®-80, or pluronic F-68) at aconcentration of about 0.01 mg/ml to about 0.5 mg/ml, e.g., about 0.01mg/ml to about 0.1 mg/ml, and optionally including a polyethylene glycol(PEG), such as a PEG molecule having a molecular weight of from about200 Daltons (Da) to about 8000 Da (e.g., about 200, 300, 400, 600, 900,1000, 1450, 3350, 4500, or 8000 Da, available from Dow Chemical). Inanother exemplary aspect, the invention provides a pharmaceuticalcomposition comprising from about 1 mg/ml to about 300 mg/ml of aCTLA-4-Ig fusion protein of the invention (e.g., D3-69-IgG2), which istypically expressed as a dimeric fusion protein, in 20 mM sodium citratebuffer in water, 100 mM NaCl, 2% by weight sucrose based on thecomposition, pH 6.5, optionally including a non-ionic detergent (e.g.,Tween®-20, Tween®-40, Tween®-60, Tween®-80, or pluronic F-68) at aconcentration of about 0.01 mg/ml to about 0.5 mg/ml, e.g., about 0.01mg/ml to about 0.1 mg/ml, and optionally including a PEG molecule, suchas a PEG molecule having a molecular weight of from about 200 Da toabout 8000 Da (e.g., about 200, 300, 400, 600, 900, 1000, 1450, 3350,4500, or 8000 Da, available from Dow Chemical).

The invention includes receptacles for containing a composition of theinvention comprising a molecule of the invention (e.g., mutant CTLA-4molecule, such as a mutant CTLA-4-Ig) and an excipient, diluent, orcarrier. The composition may be a pharmaceutical composition comprisinga molecule of the invention and a pharmaceutically acceptable excipient,diluent or carrier. Receptacles include, but are not limited to, e.g.,vials (e.g., glass vial, such as a Type I glass vial), autoinjectors,pen injectors (fixed dose or variable dose), and pre-filled syringes, orother suitable containers. If desired, a receptacle can contain one ormore pre-determined doses of the molecule of the invention effective tosuppress an immune response or treat an immune system disease ordisorder as described elsewhere herein. Some such receptacles are usefulfor administration of the composition contained therein to a subjectsuffering from an immune disease or disorder (e.g., autoinjectors, peninjectors, pre-filled syringes, etc.). Some such receptacles allow forself-administration of the composition by the subject (e.g., peninjectors, autoinjectors, pre-filled syringes, etc.).

Also provided are stable compositions or formulations of a molecule(e.g., mutant CTLA-4 ECD or mutant CTLA-4-Ig) of the invention,including pharmaceutically acceptable compositions of a molecule of theinvention with a pharmaceutically acceptable carrier. In another aspect,the invention includes freeze-dried or lyophilized compositions orformulations. The term “freeze-dried” or “lyophilized” generally refersto the state of a substance which has been subjected to a dryingprocedure such as freeze-drying or lyophilization, where at least 50% ofmoisture has been removed. Pre-lyophilization and lyophilizationprocedures are well known in the art (see, e.g., LYOPHILIZATION OFBIOPHARMACEUTICALS, Vol. 2 of BIOTECHNOLOGY: PHARMACEUTICAL ASPECTS(Henry R. Costantino et al. eds., 2004), U.S. Pat. No. 6,436,897, WO06/104852), and would be readily understood by a skilled artisan. Anysuitable lyophilization procedure can be employed or modified asappropriate by one skilled on the art in preparing the lyophilizedcomposition of the invention. A freeze-dried, air-dried, spray-dried, orlyophilized composition is usually prepared from a liquid, such as asolution, suspension, emulsion, etc. The liquid to be freeze-dried,air-dried, spray-dried, or lyophilized typically includes all of thecomponents (except the liquid, e.g., water) that are to be in the finalreconstituted liquid composition. In this way, the freeze-dried,air-dried, spray-dried, or lyophilized composition will have the desiredliquid composition (e.g., pharmaceutical composition) whenreconstituted. Exemplary compositions of the invention, includingpharmaceutical compositions, comprising a molecule of the invention(e.g., mutant CTLA-4-Ig fusion protein of the invention, such as, e.g.,SEQ ID NO:197, 199, 211, or 213), which are described throughout thisapplication, can be freeze-dried, air-dried, spray-dried, or lyophilizedto produce a stable freeze-dried, air-dried, spray-dried, or lyophilizedcomposition, respectively, by using standard procedures known in theart. See e.g., exemplary procedures described in LYOPHILIZATION OFBIOPHARMACEUTICALS, supra.

For example, a container (e.g., vial, such as a glass vial) containing aliquid composition a molecule of the invention (e.g., mutant CTLA-4-Ig,fusion protein of the invention, such as, e.g., SEQ ID NO:197, 199, 211,or 213) to be lyophilized can be lyophilized by using standardprocedures known by those of ordinary skill in the art. See, e.g.,LYOPHILIZATION OF BIOPHARMACEUTICALS, supra. The lyophilized molecule ofthe invention (e.g., mutant CTLA-4-Ig of the invention) can subsequentlybe reconstituted with a liquid to generate a reconstituted liquidcomposition. Lyophilized formulations are typically reconstituted by theaddition of an aqueous solution to dissolve the lyophilized formulation.Any suitable aqueous liquid or solution can be used to reconstitute alyophilized formulation. A lyophilized formulation is oftenreconstituted using sterile or distilled water, but solutions comprisingcarriers, excipients, diluents buffers, and/or other components,including those described throughout, can be used for reconstitution.

In one aspect, the invention provides a pharmaceutical composition inlyophilized form, wherein the composition comprises from about 1 mg/mlto about 300 mg/ml of a CTLA-4-Ig fusion protein of the invention, whichis typically expressed as a dimeric fusion protein, in appropriatebuffer (e.g., HEPES, disodium citrate-trisodium citrate, etc.) in waterat a concentration to maintain the desired pH (e.g., about pH 6.0 toabout pH 7.5), salt (e.g., 50 mM NaCl), sugar (e.g., 4-6% by weightsucrose based on the composition), and optionally including a non-ionicdetergent (Tween®-20, Tween®-40, Tween®-60, Tween®-80, or pluronic F-68)at a concentration of about 0.01 mg/ml to about 0.5 mg/ml, e.g., about0.01 mg/ml to about 0.1 mg/ml (e.g., Tween®-20, Tween®-60, Tween®-80, orpluronic F-68). A lyphophilized form of a pharmaceutical typicallyincludes a lower salt concentration and a higher sugar concentrationcompared to a non-lyophilized liquid composition.

In one particular aspect, the invention provides a stable lyophilizedcomposition for therapeutic administration upon reconstitution withsterile water which comprises a therapeutically effective amount of amolecule of the invention and optionally one or more of the followingpharmaceutically acceptable components: (a) a sugar or saccharide, sucha sucrose, mannose, dextrose, or trehalose in an amount of from about 1%by weight to about 10% by weight; (b) a detergent or emulsifier, such asTween®-20, Tween®-40, Tween®-60, Tween®-80, or pluronic F-68; (c) anisotonic agent or salt, such as an inorganic salt (e.g., sodiumchloride) in a concentration of from 0 mM to about 50 mM (including,e.g., the concentrations set forth above; (d) a suitable buffer tomaintain the pH of the composition within a range which is conducive tothe stability of the molecule; (e) dispersing agent (e.g., in an amountsufficient for long-term dispersion of the molecule of the invention,such as, e.g., from 0.001 w/v % to about 1.0 w/v %) (e.g., polysorbate,such as Tween®-20, Tween®-40, Tween®-60, or Tween®-80, or pluronicF-68); and (f) a stabilizer (e.g., saccharide, dextrans, low molecularweight (MW) PEG group, such as PEG having MW of from about 200 Da toabout 8000 Da (e.g., 200, 300, 400, 600, 900, 1000, 1450, 3350, 4500, or8000 Da) or preservative. In some such stable lyophilized compositions,the molecule of the invention is a recombinant or isolated fusionprotein of the invention, such as a fusion protein comprising apolypeptide sequence having at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to at least onepolypeptide sequence selected from the group consisting of SEQ IDNOS:74-79, 197-200, 205-214, and 219-222 (optionally, e.g., selectedfrom the group consisting of SEQ ID NOS:197, 199, 211, and 213), whereinthe fusion protein binds CD80 and/or CD86 and/or an extracellular domainthereof and/or suppresses an immune response. Such fusion proteins maybe in monomeric or dimeric form.

Exemplary amounts of each such component in the lyophilized compositioninclude those described above and herein. In one aspect, the buffer isselected so as to maintain the pH of the composition within a range offrom about pH 3 to about pH 8, from about pH 4 to about pH 7.5. Alyophilized composition of the invention comprising a recombinant mutantCTLA-4-Ig fusion protein of the invention is typically stable at −80° to+40° C. and/or substantially maintains its biological activity for atleast one week, one or more months (e.g., six months), one year, twoyears, three years, four years, or more when stored at ambienttemperatures (e.g., about 22° C. to about 30° C.). Upon reconstitutionwith a liquid (e.g., sterile water for injection (WFI)), the lyophilizedcomposition is suitable for administration (e.g., i.v., s.c.,parenteral, i.m., i.d., i.p., etc.) to a subject (e.g., human).

The invention also provides a kit comprising a lyophilized orfreeze-dried composition comprising a lyophilized or freeze-driedmolecule of the invention (e.g., mutant CTLA-4-Ig fusion protein, suchas, e.g., SEQ ID NO:197, 199, 211, or 213) in a first container (e.g.,vial, such as a glass vial) and instructions for reconstituting thefreeze-dried or lyophilized composition using a liquid (e.g., sterilewater, WFI, or buffer). Optionally, the kit further comprises a secondcontainer (e.g., vial, such as a glass vial) containing a sufficientamount of liquid (e.g., sterile water, WFI or buffer) for reconstitutionof the lyophilized or freeze-dried composition into a liquidcomposition. In this instance, reconstitution is achieved by using asyringe to remove a desired volume of water from the second containerand to introduce the water into the first container. The first containeris then rocked gently to put the molecule of the invention (e.g., fusionprotein) into solution. The kit may include a device(s) forreconstituting the lyophilized or freeze-dried composition and/oradministering the reconstituted liquid composition. Exemplary devicesinclude, but are not limited to, e.g., a two-component mixing syringe,dual-chambered syringe, and dual-chambered autoinjector. One componentor chamber contains the lyophilized composition and the second componentor chamber contains the liquid for reconstitution. With such devices,reconstitution is typically carried out just prior to administration,and the reconstituted composition is usually administered parenterally(e.g., s.c., i.v., i.m., i.d. injection).

The composition or pharmaceutical composition of the invention cancomprise or be in the form of a liposome. Suitable lipids for liposomalformulation include, without limitation, monoglycerides, diglycerides,sulfatides, lysolecithin, phospholipids, saponin, bile acids, and thelike. Preparation of such liposomal formulations is described in, e.g.,U.S. Pat. Nos. 4,837,028 and 4,737,323.

The form of the compositions or pharmaceutical composition can bedictated, at least in part, by the route of administration of thepolypeptide, conjugate, nucleic acid, vector, virus, VLP, or cell ofinterest. Because numerous routes of administration are possible, theform of the pharmaceutical composition and its components can vary. Forexample, in transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated can be included in thecomposition. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. In contrast, in transmucosaladministration can be facilitated through the use of nasal sprays orsuppositories.

A common administration form for compositions of the invention,including pharmaceutical compositions, is by injection. Injectablepharmaceutically acceptable compositions typically comprise one or moresuitable liquid carriers such as water, petroleum, physiological saline,bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.), PBS, oroils. Liquid pharmaceutical compositions can further includephysiological saline solution, dextrose (or other saccharide solution),alcohols (e.g., ethanol), polyols (polyalcohols, such as mannitol,sorbitol, etc.), or glycols, such as ethylene glycol, propylene glycol,PEG molecules, coating agents which promote proper fluidity, such aslecithin, isotonic agents, such as mannitol or sorbitol, organic esterssuch as ethyoleate, and absorption-delaying agents, such as aluminummonostearate and gelatins. The injectable composition can be in the formof a pyrogen-free, stable, aqueous solution. An injectable aqueoussolution may comprise an isotonic vehicle such as sodium chloride,Ringer's injection solution, dextrose, lactated Ringer's injectionsolution, or an equivalent delivery vehicle (e.g., sodiumchloride/dextrose injection solution). Formulations suitable forinjection by intraarticular, intravenous, intramuscular, intradermal,subdermal, intraperitoneal, and subcutaneous routes, include aqueous andnon-aqueous, isotonic sterile injection solutions, which can includesolvents, co-solvents, antioxidants, reducing agents, chelating agents,buffers, bacteriostats, antimicrobial preservatives, and solutes thatrender the formulation isotonic with the blood of the intended recipient(e.g., PBS and/or saline solutions, such as 0.1 M NaCl), and aqueous andnon-aqueous sterile suspensions that can include suspending agents,solubilizers, thickening agents, emulsifying agents, stabilizers, andpreservatives.

The administration of a polypeptide, conjugate, nucleic acid, vector,virus, pseudovirus, VLP or cell of the invention (or a compositioncomprising any such component) can be facilitated by a delivery deviceformed of any suitable material. Examples of suitable matrix materialsfor producing non-biodegradable administration devices includehydroxapatite, bioglass, aluminates, or other ceramics. In someapplications, a sequestering agent, such as carboxymethylcellulose(CMC), methylcellulose, or hydroxypropylmethylcellulose (HPMC), can beused to bind the particular component to the device for localizeddelivery.

A nucleic acid or vector of the invention can be formulated with one ormore poloxamers, polyoxyethylene/polyoxypropylene block copolymers, orother surfactants or soap-like lipophilic substances for delivery of thenucleic acid or vector to a population of cells or tissue or skin of asubject. See e.g., U.S. Pat. Nos. 6,149,922, 6,086,899, and 5,990,241.

Nucleic acids and vectors of the invention can be associated with one ormore transfection-enhancing agents. In some embodiments, a nucleic acidand/or nucleic acid vector of the invention typically is associated withone or more stability-promoting salts, carriers (e.g., PEG), and/orformulations that aid in transfection (e.g., sodium phosphate salts,dextran carriers, iron oxide carriers, or biolistic delivery (“genegun”) carriers, such as gold bead or powder carriers). See, e.g., U.S.Pat. No. 4,945,050. Additional transfection-enhancing agents includeviral particles to which the nucleic acid or nucleic acid vector can beconjugated, a calcium phosphate precipitating agent, a protease, alipase, a bipuvicaine solution, a saponin, a lipid (e.g., a chargedlipid), a liposome (e.g., a cationic liposome), a transfectionfacilitating peptide or protein-complex (e.g., a poly(ethylenimine),polylysine, or viral protein-nucleic acid complex), a virosome, or amodified cell or cell-like structure (e.g., a fusion cell).

Nucleic acids and vectors of the invention can also be delivered by invivo or ex vivo electroporation methods, including, e.g., thosedescribed in U.S. Pat. Nos. 6,110,161 and 6,261,281, and Widera et al.,J. of Immunol. 164:4635-4640 (2000).

Transdermal administration of a component of the invention (e.g.,polypeptide, conjugate, nucleic acid, vector, virus, VLP, and/or cell ofthe invention) can be facilitated by a transdermal patch comprising suchcomponent in any suitable composition in any suitable form. Suchtransdermal patch devices are provided by the invention. For example,such component can be contained in a liquid reservoir in a drugreservoir patch device, or, alternatively, the component can bedispersed throughout a material suitable for incorporation in a simplemonolithic transdermal patch device. Typically, the patch comprises animmunosuppressive amount of at least one such component—such as anamount effective to suppress an immune response in a subject contactedwith the patch. Examples of such patch devices are known in the art. Thepatch device can be either a passive device or a device capable ofiontophoretic delivery of at least one such component to the skin ortissue of the subject.

A composition, particularly a pharmaceutical composition, may compriseany suitable dose of at least one such component of the invention (e.g.,polypeptide, conjugate, nucleic acid, vector, virus, VLP, and/or cell)sufficient to achieve the desired immunosuppressive response in asubject following administration. Proper dosage can be determined by anysuitable technique and considerations for determining the proper areknown in the art. In a simple dosage testing regimen, low doses of thecomposition are administered to a test subject or system (e.g., ananimal model, cell-free system, or whole-cell assay system). Dosage iscommonly determined by the efficacy of the particular component to beadministered, the condition of the subject, the body weight of thesubject, and/or target area of the subject to be treated. The size ofthe dose is also determined by the existence, nature, and extent of anyadverse side-effects that accompany the administration of any suchparticular component in a particular subject. Principles related todosage of therapeutic and prophylactic agents are provided in, e.g.,Platt, Clin. Lab Med. 7:289-99 (1987), “Drug Dosage,” J. Kans. Med. Soc.70(1):30-32 (1969), and other references described herein (e.g.,Remington's, supra).

By way of example, a therapeutically effective amount of a polypeptideof the invention for an initial dosage for treating an autoimmunedisease may comprise from about 0.001 mg/kg body weight of the subjectto about 100 mg/kg body weight of the subject, such as, e.g., from about0.001 milligrams per kilogram (mg/kg) body weight of the subject toabout 100 milligrams per kilogram (mg/kg) weight of the subject, or,e.g., from about 0.001 mg/kg weight of the subject to at least about0.005, 0.01, 0.025, 0.05, 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80,90, or 100 mg/kg body weight of the subject. Such dosage can be by anysuitable protocol, e.g., such as administered daily, weekly, orbiweekly, or any combination thereof (e.g., at about 0, 1, 2, 4, 5, 6,and 7 days, weekly thereafter, or at about 0, 1, 2, 4, and 6 weeks),followed by 1-, 2-, 3-month intervals, and by any suitable deliverymethod, such as, e.g., by electroporation or a subcutaneous (s.c.),intramuscular (i.m.), intravenous (i.v.), or intraperitoneal (i.p.),subdermal, transdermal, parenteral, or intradermal (i.d.) injection. Insome instances, a polypeptide of the invention is typically administeredas a soluble polypeptide, such as, e.g., a fusion protein comprising amutant CTLA-4 ECD polypeptide of the invention covalently linked to anIg Fc polypeptide. For example, a pharmaceutical composition comprisinga mutant CTLA-4-Ig fusion protein of the invention in a pharmaceuticallyacceptable carrier, diluent, or excipient may be administered by anyappropriate route (e.g., intradermally, intravenously, orsubcutaneously) in an effective amount depending upon the autoimmunedisease (e.g., rheumatoid arthritis) or condition to be treated (e.g.,to inhibit rejection of a tissue, cell, graft, or solid organ transplantfrom a donor by the recipient subject).

In one exemplary aspect, the invention provides a method of suppressingan immune response in a subject in need thereof, comprisingadministering to the subject a pharmaceutical composition comprising,e.g., from about 1 mg/ml to about 300 mg/ml, including from about 25mg/ml to about 150 mg/ml (e.g., 50 or 100 mg/ml) of D3-54-IgG2 fusionprotein in 20 mM HEPES buffer in water, 100 mM NaCl, 2% by weightsucrose, pH 7.4, wherein the subject suffers from an autoimmune disorder(e.g., rheumatoid arthritis). In another exemplary aspect, the inventionprovides method of inhibiting rejection of a tissue, cell, graft orsolid organ transplant from a donor in a recipient subject, comprisingadministering to the recipient subject a pharmaceutical compositioncomprising from about 25 mg/ml to about 100 mg/ml (e.g., 50 or 100mg/ml) of D3-69-IgG2 fusion protein in 20 mM sodium citrate buffer inwater, 100 mM NaCl, 2% by weight sucrose, pH 6.5.

Also provided is a viral vector composition, which comprises a carrieror excipient and a viral vector of the invention. Pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier orexcipient and a viral vector are also provided. The amount or dosage ofviral vector particles or viral vector particle-encoding nucleic aciddepends on: (1) the type of viral vector particle with respect to originof vector, including, but not limited to, e.g., whether the vector is analphaviral vector, Semliki-Forest viral vector, adenoviral vector,adeno-associated (AAV) viral vector, flaviviral vector, papillomaviralvector, and/or herpes simplex viral (HSV) vector, (2) whether the vectoris a transgene expressing or recombinant peptide displaying vector, (3)the host, and (4) other considerations discussed above. Generally, withrespect to gene transfer vectors, the pharmaceutically acceptablecomposition comprises at least about 1×10² viral vector particles in avolume of about 1 ml (e.g., at least about 1×10² to 1×10⁸ particles inabout 1 ml). Higher dosages also can be suitable (e.g., at least about1×10⁶, about 1×10⁸, about 1×10⁹, about 1×10¹⁰ particles/ml).

The invention also provides a composition (including a pharmaceuticalcomposition) comprising an aggregate of two or more polypeptides orconjugates of the invention. Moreover, the invention provides acomposition (including a pharmaceutical composition) comprising apopulation of one or more multimeric polypeptides or multimericconjugates of the invention. As noted above, pharmaceutical compositionsinclude a pharmaceutically acceptable excipient, diluent, or carrier.

Kits

The present invention also provides kits including one, two, three, ormore of the polypeptides (e.g., mutant CTLA-4 ECD polypeptides, mutantCTLA-4-Ig fusion proteins, including dimeric fusion proteins),conjugates, nucleic acids, vectors, cells, and/or compositions of theinvention. Kits of the invention optionally comprise: (1) at least onepolypeptide (e.g., mutant CTLA-4-Ig fusion protein), conjugate, nucleicacid, vector, VLP, cell, and/or composition of the invention; (2)optionally at least one second immunosuppressive agent (e.g.,nonsteroidal anti-inflammatory agent, methotrexate, steroid, TNFαantagonist, etc.); (3) instructions for practicing any method describedherein, including a therapeutic or prophylactic method and instructionsfor using any component identified in (1) or (2); (4) a container forholding the at least one such component identified in (1) or (2); and/or(5) packaging materials. One or more of the polypeptides (e.g., mutantCTLA-4-Ig fusion protein), conjugates, nucleic acids, vectors, VLPs,cells, and/or compositions of the invention, optionally with one or moresecond immunosuppressive agents, can be packaged in packs, dispenserdevices, and kits for administration to a subject, such as a mammal,including a human. An effective amount of each such polypeptide (e.g.,mutant CTLA-4-Ig fusion protein), conjugate, nucleic acid, vector, VLP,cell, and/or composition of the invention or optional secondimmunosuppressive agent (e.g., dose) for the indicated therapeutic orprophylactic method is indicated and one or more such doses is provided.The one or more polypeptides (e.g., mutant CTLA-4-Ig fusion protein),conjugates, nucleic acids, vectors, and/or cells compositions, and, ifdesired, the optional second immunosuppressive agent, may be provided inpowder (e.g., lyophilized) or liquid form, and may be formulated with anexcipient or carrier (including, e.g., a pharmaceutically acceptableexcipient or carrier), thereby forming a composition (including, e.g.,pharmaceutical composition). Packs or dispenser devices that compriseone or more unit dosage forms are provided. Typically, instructions foradministration of such components are provided with the packaging, alongwith a suitable indication on the label that the compound is suitablefor treatment of an indicated condition.

EXAMPLES

The following examples further illustrate the invention, but should notbe construed as limiting its scope in any way.

Example 1

This example provides a description of the methods for creating aLEA29Y-Ig fusion protein, which was used as a control and forcomparative purposes in Biacore™ binding and cell-based activity assays.

Creation of DNA Plasmid Vector Encoding LEA29Y-Ig Fusion Protein.

This example describes the making of a DNA plasmid vector that encodesthe LEA29Y-Ig fusion protein. LEA29Y-Ig comprises a specific knownmutant CTLA-4 ECD polypeptide, termed “LEA29Y” (or “LEA” or “L104EA29Y”or “A29YL104E”), which is covalently linked at its C-terminus to theN-terminus of a specific mutant human IgG1 Fc polypeptide. The LEA29Ypolypeptide is a mutant CTLA-4 ECD polypeptide comprising a polypeptidesequence that differs from the polypeptide sequence of the human CTLA-4extracellular domain by two mutations—an A29Y substitution and a L104Esubstitution—where positions 29 and 104 are numbered by reference to thepolypeptide sequence of the human CTLA-4 ECD polypeptide, with the firstamino acid residue of human CTLA-4 designated as amino acid residueposition 1. See U.S. Pat. No. 7,094,874. The plasmid vectorpcDNA3.1-LEA, which includes a nucleotide sequence that encodesLEA29Y-Ig, was created to produce this fusion protein.

DNA encoding LEA29Y-Ig is created by PCR assembly using overlappingoligonucleotides designed based on sequence homology to the nucleotidesequence encoding LEA29Y-Ig shown in SEQ ID NO:167. The oligonucleotidesare designed, made, and assembled using standard procedures well knownby those of ordinary skill in the art and can include stop and startcodons and restriction sites as necessary. The PCR amplificationprocedures employed are also well known in the art. See, e.g., Berger,Ausubel, and Sambrook, all supra.

The oligonucleotides are assembled in a 100 μl PCR reaction with 1 μMoligonucleotides, 1×Taq buffer (Qiagen; #201225) and 200 μM dNTPs for 30amplification cycles (94° C., 30 s; 60° C., 30 s; 72° C., 60 s).Amplified DNA is purified by QiaQuick PCR Spin Columns (Qiagen, Cat.#28104) and digested with restriction enzymes NheI and SacII. Thefragments were separated by agarose-gel electrophoresis, purified usingQiaquick Gel Extraction Kit (Qiagen, #28704) as per manufacturer'srecommendation, and ligated into similarly digested plasmid pcDNA 3.1(+) (Invitrogen, Cat. #V790-20). Ligations are transformed into TOP10 E.coli cells (Qiagen, Cat. #C4040-10) as per manufacturer'srecommendations. The resulting cells are incubated overnight at 37° C.in LB medium containing 50 μg/ml carbenicillin with shaking at 250 rpmand then used to make a maxiprep (Qiagen; #12362) stock of plasmid DNA(referred to hereinafter as plasmid vector pcDNA3.1-LEA).

The plasmid vector pcDNA3.1-LEA is identical to the plasmid vector pcDNAmutant CTLA-4-IgG2 vector shown in FIG. 1 except that the nucleic acidsequence encoding the mutant CTLA-4-IgG2 polypeptide has been replacedby a nucleic acid sequence encoding the LEA29Y-Ig fusion protein. Anucleic acid encoding the human CTLA-4 signal peptide was included asthe signal peptide-encoding nucleotide sequence.

A nucleic acid sequence encoding the predicted LEA29Y-Ig fusion proteinis shown in SEQ ID NO:167. SEQ ID NO:167 includes the nucleotidesequence encoding the signal peptide (e.g., amino acid residues 1-37 ofSEQ ID NO:165). The polypeptide sequences of the predicted LEA29Y-Ig andmature LEA29Y-Ig fusion protein (without the signal peptide) are shownin SEQ ID NOS:165 and 166, respectively. As indicated in FIG. 2C, thepredicted amino acid sequence of LEA29Y-Ig includes the followingsegments: the predicted signal peptide (amino acid residues 1-37), theLEA29Y ECD polypeptide (amino acid residues 38-161), linker (amino acidresidue 162), and a mutant (modified) Fc domain of a human IgG1polypeptide (amino acid residues 163-394). The amino acid residues atthe junctions between these various segments are also shown in FIG. 2C.Specifically, the last four amino acid residues of the signal peptide,the first five and last five amino acid residues of the LEA29Y ECD, thesingle linker amino acid residue (Q), and the first five and last fiveamino acid residues of the mutant IgG1 Fc polypeptide are shown.

The signal peptide is typically cleaved during processing and thus thesecreted fusion protein (i.e., mature fusion protein) of LEA29Y-Ig doesnot usually contain the signal peptide sequence. The mature/secretedform of LEA29Y-Ig, which has a total of 357 amino acids, comprises aminoacid residues 38-394 (the full-length sequence without the signalpeptide) of the predicted sequence shown in SEQ ID NO:165, and beginswith the amino acid sequence: methionine-histidine-valine-alanine. SEQID NO:165 includes the signal peptide (e.g., residues 1-37) at itsN-terminus; the signal peptide is typically cleaved to form the matureprotein shown in SEQ ID NO:166. If desired, the amino acids of themature form can be numbered beginning with the Met of theMet-His-Val-Ala sequence, designating Met as the first residue (e.g.,the ECD comprises amino acid residues numbered 1-124), as in the matureLEA29Y-Ig fusion protein having the sequence shown in SEQ ID NO:166. Inone aspect, the sequence of SEQ ID NO:165 or 166 does not include theC-terminal lysine residue; this residue may cleaved during processing orprior to secretion.

The protein sequence of the LEA29Y-Ig fusion protein is described inU.S. Pat. No. 7,094,874. Specifically, SEQ ID NO:4 of U.S. Pat. No.7,094,874 shows a protein sequence encoding the non-mature form ofmonomeric LEA29Y-Ig. In U.S. Pat. No. 7,094,874, the LEA29Y-Ig fusionprotein is termed “L104EA29YIg.” The mature LEA29Y-Ig fusion proteincomprising the sequence shown in SEQ ID NO:166 set forth herein differsfrom the fusion protein sequence shown in SEQ ID NO:4 in U.S. Pat. No.7,094,874 because SEQ ID NO:4 of U.S. Pat. No. 7,094,874 includes asignal peptide (i.e., residues 1-26 of SEQ ID NO:4). This signal peptideis typically cleaved during processing and thus the mature (secreted)form of the LEA29Y-Ig fusion protein does not usually include the signalpeptide sequence. SEQ ID NO:3 of U.S. Pat. No. 7,094,874 presents anucleic acid sequence that encodes the L104EA29YIg fusion protein (i.e.,LEA29Y-Ig).

LEA29Y-Ig typically exists in solution as a dimeric fusion proteincomprising two identical monomeric fusion proteins. In this instance,each monomeric mature LEA29Y-Ig fusion protein comprises a LEA29Y ECD(SEQ ID NO:168) polypeptide fused at its C-terminus to the N-terminus ofa mutant IgG1 Fc (SEQ ID NO:186). The two LEA29Y-Ig monomers arecovalently linked together by disulfide bonds formed between cysteineresidues in each monomer, thereby forming the LEA29Y-Ig fusion proteindimer. The LEA29Y-Ig dimer is the form of the fusion protein moleculeused in the assays described in these Examples, unless explicitly statedotherwise.

Creation of Stable CHO-K1 Cell Line Expressing LEA29Y-Ig Fusion Protein.

A stable cell line was created to generate multi-milligram quantities ofthe LEA29Y-Ig fusion protein discussed above.

Transfection of CHO-K1 Cells.

CHO-K1 cells were seeded at a density of 1×10⁶ in T-175 flasks (BDFalcon, #353112) containing 40 ml Growth Medium (DMEM/F12 medium(Invitrogen, #10565-018) supplemented with 10% fetal bovine serum (FBS)(Hyclone, #SV30014.03) and 1×PS (Penicillin+Streptomycin) (Invitrogen,#15140-122)). Cells were incubated for 24 hours (hrs) at 37° C. and thentransfected with 10 μg Maxiprep plasmid DNA (e.g., plasmid vectorencoding LEA29Y-Ig as described above) mixed with 60 μl Fugene 6 (Roche,#11814443001) as per the manufacturer's recommended conditions. Cellswere incubated for 2 days (d) at 37° C. in Growth Medium and then for 10d in Selection Medium (Growth Medium containing 300 μg/ml Geneticin(Invitrogen, #10131-027), changing the media every 2 d. The medium wasremoved and cells were dispersed by addition of 3 ml 0.05% trypsin(Invitrogen, Cat. #25300-054) and incubation at 37° C. for 3 min.Dispersed cells were diluted into 10 ml Growth Medium and harvested bycentrifugation at 1000 rpm for 5 min at room temperature (RT) in aGH-3.8 rotor (Beckman Coulter, #360581). After discarding thesupernatant, cells were suspended in 1 ml growth medium, filteredthrough 40 μm membranes (BD Falcon, #352340), and adjusted to a densityof 1×10⁶ cells/ml.

Separation of Unique Clones.

Using a cell-sorter (Dako, MoFlo), live cells were individuallydispersed into 96-well culture plates (Sigma-Aldrich, #CLS-3596)containing 200 μl/well Growth Medium containing 25% Conditioned Medium(Growth Medium previously harvested from untransfected (or naïve) cellcultures). After incubation at 37° C. for 10-14 d, cells were dispersedby trypsin hydrolysis and transferred to new culture plates containing200 μl/well Growth Medium. Cells were cultured at 37° C. in GrowthMedium until cell density reached 70% confluence (approximately 14 d,with medium changed every 7 d).

Identification of Desired Clones.

Clones expressing high levels of recombinant LEA29Y-Ig fusion proteinwere identified by dot-blot and western analysis. For dot-blot analysis,100 μl of medium was harvested from each well of the 96-well cultureplates and transferred to nitrocellulose membranes (Whatman, #10439388)as per the manufacturer's recommendations. Membranes were washed twicewith 200 ml PBST (PBST is phosphate-buffered saline (PBS)+0.05%Tween®-20) for 10 min at room temperature (RT) and then incubated withPBST containing 5% nonfat dry milk (EMD, #1.15363.0500) for 1 hour (hr)at RT. Membranes were washed as described above and incubated for 1 hrat RT in 20 ml PBST containing horseradish peroxidase (HRP)-conjugatedgoat anti-human Ig antibody (Vector Labs, #BA-3000) diluted to 1:4000.Membranes were washed as described above and incubated for 1 hr at RTwith PBST containing streptavidin-HRP reagent (BD Biosciences, #554066)diluted 1:2000. Membranes were washed as described above and signalswere detected using ECL Western Blot Detection Reagent (Amersham, Cat.#RPN2132) as per the manufacturer's recommended conditions. Positiveclones identified by high signal intensity (i.e., expressing high levelsof fusion protein) were dispersed by trypsin hydrolysis and transferredto 6-well culture plates (BD Falcon, Cat. #353046) containing 2 ml/wellGrowth Medium. After incubation at 37° C. for 3-4 d, cells weredispersed by trypsin hydrolysis and transferred to T-75 flasks (BDFalcon, Cat. #353136) containing 20 ml Growth Medium. After incubationat 37° C. for 2 d, 100 μl of medium was harvested and analyzed forprotein expression levels by western analysis. For western analysis,equal amounts (15 μl) of medium from each cell culture was run through4-12% Bis-Tris NuPAGE gels (Invitrogen, #NP0322BOX) in MES (MES is2-(N-morpholino)ethanesulfonic acid, pH 7.3) running buffer (Invitrogen,#NP0002) as per the manufacturer's recommended conditions. Proteins weretransferred from gels to nitrocellulose membranes (Invitrogen, Cat.#LC2001) by electro-transfer as per the manufacturer's recommendedconditions. Membranes were processed as described above for dot-blottingand positive clones (expressing the fusion protein of interest) wereidentified by signal intensity and apparent molecular weight. Positiveclones were dispersed by trypsin hydrolysis as described above andpropagated in T-175 flasks containing 40 ml Growth Medium.

Production and Purification of LEA29Y-Ig Fusion Protein.

Propagation of Roller Bottle Cultures.

A Stable CHO-K1 cell line that had been transfected as described abovewith nucleic acid encoding the fusion protein of interest was grown toconfluence in T-175 flasks containing 40 ml Growth Medium (DMEM/F-12medium (Invitrogen, #10565-018) supplemented with 10% FBS (Hyclone#SV30014.03) and 1×PS (Invitrogen, #15140-122)). Cells were harvested byincubation in 3 ml 0.05% trypsin (Invitrogen, Cat. #25300-054) for 3 minat 37° C., diluted into 12 ml Growth Medium and then transferred toroller bottles (Corning, Cat. #431191) containing 250 ml Growth Medium.After incubation of roller bottle cultures at 37° C. in a humidifiedrolling incubator for 2 d, the media was removed and replaced with 250ml fresh Growth Medium. Cultures were incubated for 2 d at 37° C. andthe medium was replaced with 250 ml UltraCHO Medium (UltraCHO medium(BioWhittaker, Cat. #12-724) supplemented with 1/1000 EX-CYTE(Serologicals Proteins, Cat. #81129N) and 1×PS). After incubation for 2d at 37° C., the media was replaced with 250 ml fresh UltraCHO Medium.Cultures were incubated for 2 d at 37° C. and the medium was replacedwith 250 ml Production Medium (DMEM/F-12 medium supplemented with1/100×ITSA (Life Technologies #51300-044), 1/1000× EX-CYTE and 1×PS).During production, media was harvested and replaced with freshProduction Media every other day.

Protein Purification.

Production media from roller bottle cultures was clarified bycentrifugation at 2500×g for 30 min at RT followed by filtration through0.2 μM membranes (VWR, Cat. #73520-986). Media were concentrated 10-foldby tangential flow filtration using 10 kDa MWCO membranes (Millipore,Cat. #P3C010C00) and then used for Protein-A affinity chromatographyusing a BioCad vision HPLC system. Ig-fusion protein was bound to Poros20 Protein-A resin (Applied Biosystems, Cat. #1-5029-01) in PBS buffer,washed with the same buffer, eluted with 80 mM citric acid buffer (pH4.0) containing 160 mM sodium chloride and then neutralized by additionof 2M Tris base. The protein solution was finally dialyzed against 6liters (l) PBS using 10 kDa MWCO membranes (Pierce, Cat. #PI66810).

Example 2

This example describes exemplary methods used to create and screenlibraries of CTLA-4 mutants for altered human CD80 and/or human CD86binding activities by phage display.

Human CD80-Ig Fusion Protein.

Human CD80-Ig (“hCD80-Ig”) and human CD86-Ig (“hCD86-Ig”) fusionproteins were used as ligands in phage panning and phage ELISAexperiments to identify mutant CTLA-4 molecules that bind human CD80(“hCD80”) and/or human CD86 (“hCD86”) and/or an extracellular domain ofeither or both. Human CD80-Ig (also termed “hB7.1-Ig” or “hB7-1-Ig”) andhuman CD86-Ig (also termed “hB2.1-Ig” or “hB2-1-Ig”) fusion proteins areavailable from R&D Systems (Minneapolis, Minn.).

A representative nucleic acid sequence encoding the predicted WT humanCD80-IgG1 fusion protein, which comprises the human CD80 signal peptide,human CD80 ECD, and human IgG1 Fc, is shown in SEQ ID NO:172. Thepredicted and mature polypeptide sequences of the hCD80-IgG1 fusionprotein are shown in SEQ ID NO:170 and SEQ ID NO:171, respectively. Thepredicted fusion protein shown in SEQ ID NO:170 comprises WT human CD80ECD covalently fused at its C-terminus to the N-terminus of a human IgG1Fc polypeptide and includes a signal peptide at its N-terminus. Thesignal peptide is typically cleaved to form the mature CD80-Ig fusionprotein shown in SEQ ID NO:171.

Human CD80-IgG1 is typically abbreviated herein as hCD80-Ig. As shown inFIG. 2A, the predicted amino acid sequence of the hCD80-Ig fusionprotein (also designated “CD80-IgG1”) includes the following segments:the predicted signal peptide (amino acid residues 1-34), human CD80 ECD(amino acid residues 35-242), linker (amino acid residues 243-245), andhuman IgG1 Fc polypeptide (amino acid residues 246-476). The amino acidresidues at the junctions between these various segments are shown inFIG. 2A. Specifically, the last four amino acid residues of the signalpeptide, the first five and last five amino acid residues of the humanCD80 ECD, the amino acid residues of the linker (GVT), and the firstfive and last five amino acid residues of the human IgG1 Fc polypeptideare shown. In the CD80-Ig fusion protein, three residues GVT are presentas a cloning artifact (or linker) between the C-terminus of the CD80 ECD(which ends with the amino acid residues FPDN) and the N-terminus of theIgG1 Fc polypeptide (which begins with the amino acid residues PKSC).This GVT cloning artifact or linker is shown in the predicted and matureCD80-Ig polypeptide sequences shown in SEQ ID NO:170 and 171,respectively.

The signal peptide is typically cleaved during processing and thus thesecreted fusion protein (i.e., mature fusion protein) of hCD80-Ig doesnot usually contain the signal peptide. The mature/secreted form ofhCD80-Ig, which has a total of 442 amino acids, comprises amino acidresidues 35-476 (the full-length sequence without the signal peptide) ofSEQ ID NO:170, and begins with the amino acid residue sequence:valine-isoleucine-histidine-valine. If desired, the amino acids of themature form can be numbered beginning with the valine (Val) of theVal-Ile-His-Val sequence, designating Val as the first residue (e.g.,the ECD comprises amino acid residues numbered 1-208), as in the matureform of hCD80-Ig comprising the polypeptide sequence shown in SEQ IDNO:171.

The hCD80-Ig fusion protein typically exists in solution as a dimericfusion protein comprising two identical monomeric mature hCD80-Ig fusionproteins. In this instance, each monomeric mature hCD80-Ig fusionprotein (SEQ ID NO:171) comprises a human CD80 ECD (SEQ ID NO:174) fusedat its C-terminus to the N-terminus of a human IgG1 Fc (SEQ ID NO:185).The two hCD80-Ig monomers are covalently linked together by disulfidebonds formed between cysteine residues in each monomer, thereby formingthe hCD80-Ig fusion protein dimer. The hCD80-Ig fusion protein dimer isthe form of the fusion protein molecule used in the assays described inthese Examples, unless explicitly stated otherwise.

A representative nucleic acid encoding the predicted full-length humanCD80 polypeptide is shown in SEQ ID NO:196. The nucleic acid sequenceshown in SEQ ID NO:196 encodes the human CD80 signal peptide, ECD,transmembrane domain, and cytoplasmic domain, and includes the TAA stopcodon at the C-terminus.

Human CD86-Ig Fusion Protein.

A representative nucleic acid sequence encoding the predicted amino acidsequence of human CD86-human IgG1 (typically abbreviated herein as“hCD86-Ig”) fusion protein is shown in SEQ ID NO:179. This nucleic acidsequence includes a nucleotide sequence encoding a signal peptide themature human CD86-human IgG1 fusion protein. The predicted amino acidsequence of hCD86-Ig fusion protein is shown in SEQ ID NO:177, and anexemplary nucleic acid encoding the predicted hCD86-Ig fusion protein isshown in SEQ ID NO:179.

As shown in FIG. 2B, the predicted amino acid sequence of the hCD86-Igfusion protein includes the following segments: the predicted signalpeptide (amino acid residues 1-23), human CD86 extracellular domain(amino acid residues 24-243), linker sequence (amino acid residues244-246), and human IgG1 Fc polypeptide (amino acid residues 247-477).The amino acid residues at the junctions between these various segmentsare also shown in FIG. 2B. Specifically, the last four amino acidresidues of the signal peptide, the first five and last seven amino acidresidues of the human CD86 ECD, the amino acid residues of the linker(GVT), and the first five and last five amino acid residues of the humanIgG1 Fc polypeptide are shown.

The CD86 signal peptide is typically cleaved from the predicted hCD86-Igpolypeptide during processing and thus the secreted human CD86-Ig fusionprotein (i.e., mature fusion protein) does not usually include thesignal peptide. The mature/secreted form of hCD86-Ig, which has a totalof 454 amino acids, comprises amino acid residues 24-477 (thefull-length sequence without the signal peptide) of SEQ ID NO:177, andbegins with the following amino acid residue sequence:alanine-proline-leucine. If desired, the amino acids of the maturefusion protein can be numbered beginning with the alanine residue (Ala)of the Ala-Pro-Leu sequence, designating Ala as the first residue (e.g.,the ECD comprises amino acid residues numbered 1-218), as in the matureform of hCD86-Ig comprising the polypeptide sequence shown in SEQ IDNO:178. The mature fusion protein (SEQ ID NO:178) comprises a WT hCD86ECD protein covalently fused at its C-terminus to the N-terminus of ahIgG1 Fc polypeptide.

The hCD86-Ig fusion protein typically exists in solution as a dimericfusion protein comprising two identical monomeric mature hCD86-Ig fusionproteins. In this instance, each monomeric mature CD86-Ig fusion protein(SEQ ID NO:178) comprises a human CD86 ECD (SEQ ID NO:180) fused at itsC-terminus to the N-terminus of a human IgG1 Fc (SEQ ID NO:185). The twohCD86-Ig monomers are covalently linked together by disulfide bondsformed between cysteine residues in each monomer, thereby forming thehCD86-Ig fusion protein dimer. The hCD86-Ig fusion protein dimer is theform of the fusion protein molecule used in the assays described inthese Examples, unless explicitly stated otherwise.

In the CD86-Ig fusion protein (e.g., predicted and mature forms), threeresidues GVT are present as a cloning artifact (or linker) between theC-terminus of the CD86 ECD and the N-terminus of the IgG1 Fcpolypeptide. In another aspect, the WT human CD86 ECD protein comprisesa polypeptide sequence comprising amino acid residues 1-218 of SEQ IDNO:180 (i.e., excluding the last two C-terminal amino acid residues atthe (PP)).

Orencia® Fusion Protein.

As an additional control and for comparative purposes, a commerciallyavailable fusion protein known as the Orencia® fusion protein(Bristol-Myers Squibb Co., Princeton, N.J.) was purchased. The Orencia®fusion protein is composed of the WT human CTLA-4 extracellular domaincovalently fused at its C-terminus to the N-terminus of a specificmutant IgG1 Fc polypeptide. The Orencia® protein is a dimeric fusionprotein comprising two identical monomeric fusion proteins covalentlylinked together by disulfide bonds formed between cysteine residuespresent in each monomeric fusion protein. The polypeptide sequence ofeach mature Orencia® fusion protein monomer is shown in SEQ ID NO:164and is composed of the following segments: a WT human CTLA-4extracellular domain (amino acid residues 1-124), linker sequence (aminoacid residue 125), and a mutant IgG1 Fc polypeptide (amino acid residues126-357). Each Orencia® fusion protein monomer has a structure similarto that of the LEA29Y-Ig fusion protein monomer shown schematically inFIG. 2C, except that the LEA29Y ECD is replaced with WT human CTLA-4ECD, and no signal peptide is present in either Orencia® fusion proteinmonomer, because each monomer is a secreted or mature fusion protein.The polypeptide sequence of the non-mature form of an Orencia® monomer(which includes a signal peptide) and a nucleic acid sequence encodingthe non-mature form of an Orencia® fusion protein monomer are shown inSEQ ID NO:8 and SEQ ID NO:7, respectively, of U.S. Pat. No. 7,094,874.Methods of making and using the Orencia® fusion protein are alsodisclosed in U.S. Pat. No. 7,094,874.

Creation of DNA Sequences Encoding Mutant CTLA-4 Polypeptides.

Directed evolution methods were used to generate libraries ofrecombinant non-naturally-occurring polynucleotides encoding recombinantmutant CTLA-4 extracellular domain polypeptides. The protein andnucleotide sequences of a number of naturally-occurring mammalian CTLA-4homologues are known. See, e.g., National Center for BiotechnologyInformation (NCBI). Sequence diversity identified in a variety ofnaturally-occurring mammalian CTLA-4 extracellular domain homologues wasused in directed evolution methods to generate libraries of recombinantpolynucleotides encoding mutant CTLA-4 ECD domain polypeptides. Directedevolution procedures include, e.g., in vitro recombination andmutagenesis procedures as substantially described in Stemmer, Proc.Natl. Acad. Sci. USA 91:10747-10751 (1994); Chang et al., Nature Biotech17:793-797 (1999); Int'l Pat. Appn. Publ. No. WO 98/27230; and U.S. Pat.Nos. 6,117,679 and 6,537,776.

Creation of Libraries of DNA Sequences Encoding Mutant CTLA-4Polypeptides.

Mutant DNA sequences encoding recombinant mutant CTLA-4 ECD polypeptideswere amplified from assembly reactions by PCR using forward and reverseprimers designed based on sequence homology. The primers were designed,made, and assembled using standard procedures well known by those ofordinary skill in the art and included stop and start codons andrestriction sites as necessary. The PCR amplification proceduresemployed are also well known in the art. See, e.g., Berger, Ausubel, andSambrook, all supra. Exemplary forward and reverse primers include, butare not limited, to the following: forward primer(5′-CTATTGCTACGGCCGCTATGGCCMTKCACGTCGCTCAACCAGCCGTCGTACTC GCGTCC-3′)(SEQ ID NO:191) and reverse primer(5′-GTGATGGTGATGGTGTGCGGCCGCATCAGA-3′) (SEQ ID NO:192). 5 μl of assemblyreaction was used as template in a 100 μl PCR reaction with 1 μM forwardand reverse primers, Taq buffer (Qiagen; #201225) and 200 μM dNTPs for15 amplification cycles (94° C. 30 s; 50° C. 30 s; 72° C. 40 s).Amplified DNA encoding mutant CTLA-4 ECD polypeptides was digested withrestriction enzymes (SfiI and NotI) and the fragments were separated byagarose-gel electrophoresis, purified using the Qiaquick Gel ExtractionKit (Qiagen, #28704) as per manufacturer's recommendation, and ligatedinto similarly digested phage-display vector pSB0124 (procedure similarto that described in Chang et al., Nature Biotech 17:793-797 (1999)).The resulting library ligation was transformed by electroporation intoTOP10 E. coli cells (Invitrogen, Inc; #C4040-50) following themanufacturer's recommend conditions. Transformed cells were incubated inLB (Luria broth media) containing 50 μg/ml carbenicillin at 250 rpmovernight at 37° C. and then used to make a maxiprep (Qiagen; #12362)stock of library DNA as per the manufacturer's recommended conditions.

Screening of Phage Libraries Displaying Mutant CTLA-4 PolypeptidesHaving Improved Human CD80 and/or Human CD86 Binding Avidities

Generation of Phage Displaying Libraries of Mutant CTLA-4 Polypeptides.

Library DNA (e.g., a library of DNA sequences encoding CTLA-4 ECDmutants) was transformed by electroporation into TG-1 E. coli cells(Stratagene; #200123) as per the manufacturer's recommended conditions.The culture was grown under phagemid-selection conditions (LB mediumcontaining carbenicillin at 50 μg/ml) for 1-2 generations, infected withhelper phage M13KO7 (at a multiplicity of infection level of 5-10), andincubated with shaking at 250 rpm overnight at 37° C. under dualselection for phagemid (carbenicillin at 50 μg/ml) and helper phage(kanamycin at 70 μg/ml). The cultures were clarified by centrifugation(6000 rpm, 15 min, 4° C. in a Sorvall 600TC rotor) and phage particleswere precipitated by incubating 32 ml culture supernatant with 8 mlPEG/NaCl solution (20% PEG-8000; 2.5 M NaCl) on ice for 30 min followedby centrifugation (9500 rpm, 40 min, 4° C. in a Sorvall 600TC rotor).The phage pellet was suspended in 1 ml PBS containing 1% BSA (bovineserum albumin, Sigma; #A7906), transferred to a microfuge tube, andclarified by centrifugation (max speed, 5 min, RT in an Eppendorftable-top centrifuge).

Panning of CTLA-4 Mutant Phage Libraries.

Phage libraries were panned in up to five rounds against hCD80-Ig orhCD86-Ig fusion proteins using standard conditions. See, e.g., Lowman,et al., Biochemistry 12; 30(45):10832-10838 (1991); Smith, G. P. et al.,Chem. Rev. 97:391-410 (1997). Each round of panning included: (a)binding of phage displaying mutant CTLA-4 ECD polypeptides to hCD80-Igor hCD86-Ig ligands; (b) removal of unbound phage; (c) elution of boundphage; and (d) amplification of eluted phage for the next round ofpanning. An aliquot of phage from each round was transduced into E. colicells to obtain individual transductant colonies.

Identification of CTLA-4 Mutants Having Improved Binding Avidities toHuman CD80 and/or Human CD86 by Phage ELISA.

Individual colonies obtained from each round of panning were inoculatedinto 96-well culture plates (NUNC; #243656) containing 150 μl/well of2×YT (yeast-tryptone) media containing 50 μg/ml carbenicillin andincubated at 250 rpm overnight at 37° C. The overnight cultures wereused to inoculate deepwell blocks (Scienceware; #378600001) containing600 μl/well of the same media. Cultures were incubated at 250 rpm for 2hrs at 37° C., infected with M13K07 helper phage (multiplicity ofinfection (moi) 5-10) and then incubated at 250 rpm at overnight at 37°C. under dual selection for phagemid and helper phage markers(carbenicillin at 50 μg/ml and kanamycin at 70 μg/ml, respectively).Cultures were clarified by centrifugation at 4000 rpm for 20 min at 4°C. in a Beckman GH 3.8 rotor. ELISA plates (NUNC; #449824) were coatedby addition of 50 μl/well PBS containing 10 μg/ml hCD80-Ig or hCD86-Igand incubated overnight at 4° C. Plates were washed three times with 200μl/well PBST and blocked by addition of 200 μl/well PBS containing 3%non-fat dry milk and incubation at RT for 1 hour (hr). 25 μl/well ofphage supernatants from the deepwell block was transferred to ELISAplates containing 25 μl/well 6% non-fat dry milk and plates wereincubated for 1 hr at room temperature (RT). Plates were washed threetimes with 200 μl/well PBST and incubated with 50 μl/well HRP-conjugatedanti-M13 monoclonal antibody (GE Healthcare, #27-9421-01) diluted 1:5000in PBST containing 3% non-fat dry milk for 1 hr at RT. Plates werewashed three times with 200 μl/well PBST and signal was detected using aTMB substrate kit (Pierce; #34021) according to manufacturer'srecommended conditions. Mutant CTLA-4 ECD polypeptides exhibitingincreased binding avidity to human CD80 and/or human CD86 (as measuredby an increased avidity to hCD80-Ig and/or hCD86-Ig), compared to thebinding avidity of human CTLA-4 to human CD80 and/or human CD86 (asmeasured by the binding avidity of human CTLA-4 ECD to hCD80-Ig and/orhCD86-Ig), were selected for further analysis.

Example 3 Creation of an IgG2 Fc Fusion Protein Vector

A plasmid IgG2-Fc fusion protein expression vector was created toproduce a fusion protein comprising a mutant CTLA-4 ECD polypeptide ofthe invention and human IgG2 Fc polypeptide. DNA encoding the human IgG2Fc polypeptide was generated by PCR amplification of human leukocytecDNA (BD Biosciences, Cat. #HL4050AH) using forward primer(5′-AAGCTGTCACCGGTGGATCGATCCCGAACCCTGCCCTGATTCTGATGAGCGCAAATGTTGTGTCGAGTGCCCACCGT-3′) (SEQ ID NO:189) and reverseprimer (5′-CAGAATTCATTATTTACCCGGAGACAGGGAGAGGCT CTTCTG-3′) (SEQ IDNO:190). The primers were designed, made, and assembled using standardtechniques well known by those of ordinary skill in the art and includedstop and start codons and restriction sites as necessary. The PCRamplification procedures employed are also well known in the art. See,e.g., Berger, Ausubel, and Sambrook, all supra. 50-100 ng of cDNA wasused as template in a 100 μl PCR reaction with 1 μM forward and reverseprimers, Taq buffer (Stratagene; #200435) and 200 μM dNTPs for 25amplification cycles (94° C., 30 s; 55° C., 30 s; 72° C., 60 s). The PCRproduct was purified by QiaQuick PCR Spin Columns (Qiagen #28106) as perthe manufacturer's recommended conditions and digested with restrictionenzymes AgeI and EcoRI. The PCR digestion fragment was separated byagarose-gel electrophoresis and purified using Qiaquick Gel ExtractionKit (Qiagen, #28704) as per the manufacturer's recommended conditions. Amodified version of the pcDNA3.1-LEA vector (described above), whichcontains an AgeI restriction site in the CTLA-4 signal sequence(introduced as a silent mutation), may be digested with AgeI and EcoRIand ligated to the fragment mentioned above. The ligation is transformedinto One-shot TOP10 E. coli cells (Invitrogen Cat. #C4040-03) as per themanufacturer's recommended conditions. Transformed cells are incubatedin LB (Luria broth media) containing 50 μg/ml carbenicillin at 250 rpmovernight at 37° C. and then used to make a maxiprep (Qiagen; #12362)stock of plasmid DNA as per the manufacturer's recommended conditions.

The resulting plasmid expression vector is designated as the pcDNA IgG2Fc fusion protein expression vector. This vector is identical to thepcDNA mutant CTLA-4-IgG2 plasmid vector shown in FIG. 1 except that thenucleic acid sequence encoding the mutant CTLA-4 ECD polypeptide isremoved. The nucleic acid sequence encoding the signal peptide in pcDNAIgG2 Fc fusion vector (which in FIG. 1 can be any suitable signalpeptide-encoding nucleotide sequence) is a nucleic acid encoding thehuman CTLA-4 signal peptide (SEQ ID NO:181 or SEQ ID NO:215). This IgG2Fc fusion vector does not include any nucleic acid encoding a mutantCTLA-4 ECD polypeptide of the invention or any other CTLA-4 ECDpolypeptide.

Cloning of Nucleotide Sequences Encoding Mutant CTLA-4 Polypeptides intothe IgG2 Fc Fusion Vector.

To produce mutant CTLA-4 polypeptides as soluble Fc fusion proteins, DNAsequences encoding mutant CTLA-4 ECD polypeptides identified as havingimproved binding avidities to human CD80 and/or human CD86 (as comparedto the binding avidity of human CTLA-4-ECD to human CD80 and/or humanCD86) from the phage library screening were each cloned into the IgG2 Fcfusion protein vector described above using, e.g., the followingprocedure.

DNA sequences encoding mutant CTLA-4 ECD polypeptides exhibiting higherbinding avidities to hCD80-Ig and/or hCD86-Ig were first recovered fromthe phage display vector by PCR amplification using forward and reverseprimers designed based on sequence homology to a number of nucleotideresidues (e.g., 30-60 nucleotides) at the N- and C-termini of the mutantCTLA-4 ECD polypeptides and standard procedures known in the art. See,e.g., procedures described in, e.g., Berger, Ausubel, and Sambrook, allsupra. For example, in one exemplary aspect, DNA sequences encodingmutant CTLA-4 ECD polypeptides were recovered from the phage displayvector by PCR amplification using forward primer(5′-GGAATACCGGTTTTTTGTAAAGCCATGCACGTCG CTCAACCAGCCGTCGTACTC-3′) (SEQ IDNO:191) and reverse primer (5′-GGCACTCAGATCTACGTCATCGATCCCGAA-3′) (SEQID NO:192). 10 nanograms (ng) of plasmid DNA (phage display vectorcontaining a mutant CTLA-4 ECD-encoding nucleotide sequence) was used astemplate in a 100 μl PCR reaction with 1 μM forward and reverse primersdescribed above, Taq buffer (Stratagene; #200435) and 200 μM dNTPs for25 amplification cycles (94° C., 30 s; 55° C., 30 s; 72° C., 60 s). ThePCR product was purified by QiaQuick PCR Spin Columns (Qiagen #28106) asper the manufacturer's recommended conditions and digested withrestriction enzymes AgeI and ClaI. The fragments were separated byagarose-gel electrophoresis, purified using Qiaquick Gel Extraction Kit(Qiagen, #28704) as per the manufacturer's recommended conditions, andligated into similarly digested plasmid IgG2 Fc Fusion vector. Theligation was transformed into One-shot TOP10 E. coli cells (InvitrogenCat. #C4040-03) as per the manufacturer's recommended conditions.Transformed cells were incubated in LB (Luria broth media) containing 50μg/ml carbenicillin at 250 rpm overnight at 37° C. and then used to makea maxiprep (Qiagen; #12362) stock of plasmid DNA as per themanufacturer's recommended conditions.

The resulting plasmid expression vector, which comprises a nucleic acidencoding a mutant CTLA-4-IgG2 fusion protein of the invention, isdesignated as the pcDNA mutant CTLA-4 ECD IgG2 Fc plasmid expressionvector. A schematic diagram of this vector is shown in FIG. 1. Thisvector includes a Bla promoter; ampicillin resistant gene; pUC origin;SV40 polyadenylation (poly A) signal sequence; f1 origin; SV40 promoter;neomycin resistance gene; CMV promoter to facilitate expression of amutant CTLA-4-Ig fusion protein of the invention (comprising, e.g.,human CTLA-4 signal peptide, mutant CTLA-4 ECD polypeptide, and humanIgG2 Fc polypeptide); a nucleic acid sequence encoding a human CTLA-4signal peptide (SEQ ID NO:181 or SEQ ID NO:215); a nucleic acid sequenceencoding a mutant CTLA-4 ECD polypeptide of the invention (including,but not limited to, e.g., a nucleotide sequence encoding any one of SEQID NOS:80-152); an exemplary nucleic acid sequence encoding a human IgG2Fc polypeptide is shown in SEQ ID NO:183 or SEQ ID NO:217; and a bovinegrowth hormone (bGH) poly A termination signal sequence. The nucleicacid sequence of SEQ ID NO:183 encodes the hIgG2 Fc polypeptide with theC-terminal lysine (K) residue (SEQ ID NO:184); the nucleic acid sequenceof SEQ ID NO:217 encodes the hIgG2 Fc polypeptide without the C-terminallysine residue (SEQ ID NO:218).

The plasmid IgG2 Fc fusion protein vector can also be used to produce ahuman CTLA-4-IgG2 (“hCTLA-4-Ig”) fusion protein. In this instance, anucleic acid sequence encoding human CTLA-4 ECD (e.g., SEQ ID NO:193) iscloned into the plasmid IgG2 Fc fusion protein vector using standardcloning procedures similar to those described above in place of thenucleotide sequence encoding the mutant CTLA-4 ECD. The hCTLA-4-Igfusion protein typically exists in solution as a dimeric fusion proteincomprising two identical monomeric mature hCTLA-4-Ig fusion proteins. Inthis instance, each monomeric mature hCTLA-4-Ig fusion protein comprisesa human CTLA-4 ECD (SEQ ID NO:159) fused at its C-terminus to theN-terminus of a human IgG2 Fc (SEQ ID NO:218 or SEQ ID NO:184). The twohCTLA-4-Ig monomers are covalently linked together by disulfide bondsformed between cysteine residues in each monomer, thereby forming thehCTLA-4-Ig fusion protein dimer. The mature hCTLA-4-Ig fusion proteindimer is the form of the fusion protein used in the assays described inthese Examples, unless explicitly stated otherwise.

We have found experimentally that a human CTLA-4-Ig fusion protein ormutant CTLA-4-Ig fusion protein made in CHO cells by transfecting anexpression vector comprising a nucleotide sequence encoding thehCTLA-4-Ig or mutant CTLA-4-Ig fusion protein and the hIgG2 Fcpolypeptide shown in SEQ ID NO:184 does not typically include thepredicted C-terminal lysine (K) residue, as determined by LCMS analysis;thus, the hIgG2 Fc polypeptide sequence of a hCTLA-4-IgG2 or a mutantCTLA-4-IgG2 is that shown in SEQ ID NO:218, which hIgG2 Fc polypeptidesequence does not typically include the C-terminal lysine residue ascompared to the polypeptide sequence shown in SEQ ID NO:184.

Transient Transfection of COS Cells.

COS-7 cells were grown to 80-90% confluence in T-175 flasks containing40 ml Growth Medium (DMEM/F12 medium (Invitrogen, Cat. #10565-018)supplemented with 10% FBS (Hyclone Cat. #SV30014.03) and 1×PSG(penicillin, streptomycin and glutamine) (Invitrogen, Cat. #10378-016)).Immediately prior to transfection of the cells with a plasmid expressionvector, the medium was removed and replaced with 35 ml Expression Medium(OptiMem media (Gibco #51985) containing 1×PSG). Plasmid DNA (10 μg)(e.g., a pcDNA mutant CTLA-4 ECD IgG2 Fc expression vector encoding amutant CTLA-4-IgG2 fusion protein of the invention) was mixed withFuGENE6 transfection reagent (Roche #11 815 075 001) in a 1:3 volumeratio and added to 1 ml of Growth Medium. This mixture was then addedslowly to the T-175 flask and swirled gently to mix. After incubation at37° C. for 3 days, the media was harvested, fresh Expression Medium wasadded, and the cultures were incubated for an additional 3 d.

A similar procedure can be used to make COS-7 cells transfected with asimilar plasmid vector encoding human CTLA-4-IgG2 or a pcDNA3.1-LEAplasmid vector encoding LEA29Y-Ig.

Purification of Proteins.

Supernatants from transfection cultures (e.g., comprising cellstransfected with a pcDNA mutant CTLA-4 ECD IgG2 Fc vector encodingmutant CTLA-4-Ig fusion protein) were clarified by centrifugation at1000×g for 10 min at RT and filtration through 0.2 μm membranes(Nalgene, VWR #73520-982). Proteins were purified by Protein-A affinitychromatography using an AKTA Explorer HPLC system (GE Healthcare). Amutant CTLA-4-Ig fusion protein was bound to Hitrap Protein A FF columns(GE Healthcare, #17-5079-01) in PBS buffer, washed with the same buffer,eluted with 100 mM citric acid buffer (pH 4.0), and then neutralized byaddition of 1/10 volume of 2M Tris base. The buffer in the proteinsolution was finally exchanged to PBS by dialysis using 10 kDa MWCOmembranes (Pierce, Cat. #PI66810).

A similar procedure can be used to purify human CTLA-4-IgG2 or LEA29Y-Igfusion proteins.

Evaluation of Protein Quality.

SDS/PAGE Analysis.

The apparent molecular weight (MW) of a purified mutant CTLA-4-Ig fusionprotein of the invention was measured by SDS/PAGE analysis undernon-reducing conditions. Under non-reducing conditions, a mutantCTLA-4-Ig fusion protein of the invention typically exists as a dimericfusion protein comprising two monomeric mutant CTLA-4-Ig fusionproteins. In one aspect, the dimer is a homodimer comprising twoidentical mutant CTLA-4-Ig fusion protein monomers. In one aspect, eachCTLA-4-Ig fusion protein monomer comprises a mature/secreted mutantCTLA-4 ECD fused at its C-terminus to the N-terminus of a human IgG2 Fcpolypeptide. The two mutant CTLA-4-Ig monomers are covalently linkedtogether by disulfide bonds formed between cysteine residues in eachmonomer. The homodimer is the form of the mutant fusion protein moleculeof the invention typically described in these Examples, unlessexplicitly stated otherwise. The data shown in these Examples pertain tomutant CTLA-4-Ig fusion protein homodimers, unless explicitly statedotherwise.

SDS/PAGE analyses were performed as follows. 2 μg of purified proteinwas added to 20 μl LDS Sample Buffer (Invitrogen #NP0007) and runthrough NuPAGE 4-12% Bis-Tris gels (Invitrogen #NP0321BOX) in 1×MESsodium docecyl sulfate (SDS)/PAGE running buffer (Invitrogen #NP0002)following the manufacturer's recommended conditions. Gels were stainedby incubation in 50 ml SimplyBlue SafeStain (Invitrogen #LC6060) for 1hr with gentle agitation at RT. Gels were de-stained by two incubationswith 200 ml water for 1 hr with gentle agitation at RT and processed indrying buffer (Bio RAD 161-0752) according to the manufacturer'srecommended conditions.

A representative SDS/PAGE gel of two exemplary mutant CTLA-4-Ig fusionprotein homodimers of the invention (designated as clones D3 and D4) andthe Orencia® fusion protein, which serves as a comparative control, ispresented in FIG. 3. The D3-Ig fusion protein dimer comprises twoidentical D3-Ig fusion protein monomers that are covalently linked bydisulfide bonds formed between cysteine residues in each D3-Ig monomer.Each D3-Ig monomer comprises a mutant CTLA-4 ECD polypeptide (named“D3”) comprising the polypeptide sequence shown in SEQ ID NO:61 fuseddirectly (i.e., with no “linker” amino acid residue(s)) at itsC-terminus to the N-terminus of the hIgG2 Fc polypeptide shown in SEQ IDNO:218. The D4-Ig fusion protein dimer similarly comprises two identicalD4-Ig monomers that are covalently linked by disulfide bonds formedbetween cysteine residues in each D3-Ig monomer. Each monomeric D4-IgG2fusion protein comprises a mutant CTLA-4 ECD polypeptide (named “D4”)comprising the polypeptide sequence shown in SEQ ID NO:62 fused directly(i.e., with no “linker” amino acid residue(s)) at its C-terminus to theN-terminus of the hIgG2 Fc polypeptide sequence shown in SEQ ID NO:218.

As explained above, we have found experimentally that a mutant CTLA-4-Igfusion protein made in CHO cells using a vector comprising a nucleotidesequence encoding the predicted hIgG2 Fc polypeptide shown in SEQ IDNO:184 does not typically include the predicted C-terminal lysine (K)residue, as determined by LCMS analysis; thus, the hIgG2 Fc polypeptidesequence of a mutant CTLA-4-IgG2 described herein is that shown in SEQID NO:218, which hIgG2 Fc polypeptide sequence does not typicallyinclude the C-terminal lysine residue, as compared to the polypeptidesequence shown in SEQ ID NO:184.

SDS/PAGE analyses were performed on all protein preparations to verifyprotein quality in terms of apparent molecular weight, proteinconcentration, and purity. The results of the SDS/PAGE analyses weresimilar for all protein preparations. From the exemplary gel resultsshown in FIG. 3, purified D3-IgG2 and D4-IgG2 fusion protein dimers havean apparent MW of approximately 80 kDa, which is consistent with thepredicted MW of the exemplary homodimeric mutant CTLA-4-IgG2 fusionprotein structure depicted in FIG. 10 (predicted MW=78-79 kDa).(Purified mutant CTLA-4-IgG2 protein monomers typically have apparentMWs of 39-40 kDa.) The protein bands in the gel shown in FIG. 3 havebeen stained with equivalent intensities; this confirms the accuracy ofmeasuring protein concentration for different samples. As for proteinpurity, a lower MW band can be observed in FIG. 3 at an apparent MW ofapproximately 44 kDa, which is consistent with the predicted MW of amonomeric IgG2 fusion protein. The relative intensity of this lower MWband is consistently low and is estimated to be less than 5% of thetotal protein.

Analogous SDS/PAGE analyses can be used to evaluate the purity of humanCTLA-4-IgG2 or LEA29Y-Ig fusion proteins produced using methods similarto those describe above.

Endotoxin Analysis.

Endotoxin levels of mutant CTLA-4 fusion proteins were measured using aQCL-1000 limulus amoebocyte lysate assay kit (Cambrex #50-648U)following the manufacturers recommended conditions. The maximumendotoxin level for proteins used in cell-based assays was set at 10endotoxin units (EU)/mg protein.

Analogous analyses can be used to measure the endotoxin levels of humanCTLA-4-IgG2 or LEA29Y-Ig protein preparations.

Size Exclusion Chromatography (SEC) Analysis.

Protein aggregation levels (including aggregation levels of mutantCTLA-4 polypeptides and control polypeptides) were measured by sizeexclusion chromatography using a Dionex BioLC system (Dionex). Protein(5 μg) was run through a Tosoh G3000Wx1 column (Tosoh Bioscience) in PBSrunning buffer using a 20 min isocratic run and detection by absorbance(A) at 214 nanometers (nm). The maximum level of aggregation for proteinused in further assays was set at 10%.

SEC analysis was performed on all protein preparations to verify proteinquality in terms of protein aggregation levels. The results were similarfor all protein preparations and a representative elution profile froman SEC analysis of a mutant CTLA-4-Ig fusion protein dimer (D3-Ig) isshown in FIG. 4. The y-axis shows milliAbsorbance units (mAU); thex-axis shows elution time in minutes. The structure of the D3-Ig dimeris explained above in the “SDS/PAGE Analyses” section. This purifiedmutant CTLA-4-Ig dimer is largely homogeneous in size and does notcontain high levels of aggregated species. Other mutant CTLA-4-Ig fusionproteins of the invention were similarly analyzed and showed similarresults (data not shown). Purified mutant CTLA-4-Ig fusion proteins ofthe invention were found to be homogenous in size and did not containhigh levels (>10%) of aggregated proteins. It is important to verify theaggregation states of the purified mutant CTLA-4-Ig fusion proteins ofthe invention because highly aggregated mutant CTLA-4-Ig fusion proteinsmay bind with higher avidity to human CD80 and/or human CD86 moleculesand thus may exhibit higher biological activity.

Example 4 Measuring Binding Avidities of Mutant CTLA-4-Ig FusionProteins to Human CD80-Murine Ig (hCD80-mIg) and Human CD86-Murine Ig(hCD86-mIg) Fusion Proteins Using Surface Plasmon Resonance (SPR)Biacore™ Analysis

This example describes a procedure for screening mutant CTLA-4-Ig fusionproteins for improved binding avidity to hCD80-mIg and/or hCD86-mIgligands using a Biacore interaction analysis. In the nomenclature usedto describe this type of analysis, the immobilized binding partner isreferred to as the “ligand”, and the binding partner in the mobile phaseis referred to as the “analyte”. Fusion proteins containing an Ig domaintypically form dimeric structures in solution by virtue of strongassociation between two Ig domains. Unless indicated otherwise, suchdimeric conformations are expected to exist for the fusion proteinsdescribed in this Example (i.e., mutant CTLA-4-Ig, Orencia® fusionprotein, LEA29Y-Ig, hCD80-mIg, and hCD86-mIg). The term “avidity”typically relates to the strength of binding between dimeric analytes(e.g., mutant CTLA-4-Ig fusion proteins) and dimeric ligands (e.g.,hCD80-mIg or hCD86-mIg fusion proteins). The increases in bindingavidity described for mutant CTLA-4-Ig fusion proteins result fromincreases in binding affinity between each CTLA-4 ECD domain and itscorresponding ligand. The strength of binding avidity is typicallydescribed in terms of the equilibrium dissociation constant (K_(D)),which describes the molar concentration of analyte at which 50% ofavailable ligand is bound at equilibrium.

In this screening method, Biacore sensor chips were derivatized withhCD80-mIg or hCD86-mIg ligands and mutant CTLA-4-Ig fusion proteins inbuffer were allowed to flow over the ligand-coated sensor chips. Theability of a mutant CTLA-4-Ig fusion protein molecule to bind to aspecific binding partner (i.e., hCD80-mIg or hCD86-mIg) was evaluated.Control fusion proteins (i.e., human CTLA-4-IgG2 fusion protein,Orencia® fusion protein, and mutant LEA29Y-Ig fusion protein) were alsoallowed to flow over the ligand-coated sensor chips and the abilities ofthese molecules to bind to hCD80-mIg or hCD86-mIg were similarlyevaluated for comparison. Using the Biacore system, the association(k_(on)) and dissociation (k_(off)) rate constant of a protein ofinterest binding to hCD80-mIg or hCD86-mIg ligands can be evaluated andused to calculate the equilibrium dissociation constant, K_(D). Thehuman CTLA-4-IgG2 fusion protein, which comprises the WT human CTLA-4ECD polypeptide fused to the human IgG2 polypeptide, can serve as awild-type human CTLA-4-Ig “control”. In addition or alternatively,because the Orencia® fusion protein is composed of the WT human CTLA-4ECD polypeptide fused to a modified IgG1 Fc polypeptide, it alsoeffectively serves as a wild-type human CTLA-4-Ig control forcomparative purposes. Mutant CTLA-4-Ig fusion proteins having increasedbinding avidities for hCD80-mIg and/or hCD86-mIg, compared to humanCTLA-4-IgG2, Orencia® fusion protein, and/or LEA29Y-Ig, were identified.As discussed in greater detail below, the increased binding avidity of amutant CTLA-4-Ig fusion protein of the invention for hCD80-mIg and/orhCD86-mIg, as compared to the binding avidity of Orencia® fusion protein(i.e., hCTLA-4-IgG1) and/or LEA29Y-Ig for hCD80-mIg and/or hCD86-mIg,was not due to differences between IgG2 present in the mutant CTLA-4-Igmolecules and the mutant IgG1 present in the Orencia® or LEA29Y-Igmolecules.

All Biacore™ analyses were performed on a Biacore™ 2000 system (GEHealthcare) at room temperature (RT, 25° C.). HBS-EP buffer (10 mM HEPES(pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) was used as theflow buffer for all experiments.

Standard Kinetic Assay.

The standard kinetic assay measures binding kinetics of dimeric ligand(e.g., hCD80-mIg fusion protein or hCD86-mIg fusion protein) coated tosensor chips and dimeric analytes (e.g., mutant CTLA-4-Ig fusionproteins of the invention) in the mobile phase. Rabbit anti-mouse IgGantibody (GE Healthcare, #BR-1005-14) was immobilized on CM5 sensorchips (GE Healthcare, #BR-1000-14) according to the manufacturer'sprotocol. Antibody was diluted to 30 μg/ml in immobilization buffer (10mM sodium acetate, pH 5.0 (BR-1003-51)). At a flow rate of 5 μl/minute,sensor chip CM5 was activated with a 35 μl injection of a mixture of1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) (made by mixing equal volumes of 11.5 mg/mlEDC and 75 mg/ml NHS) (GE Healthcare, #BR-1000-50), followed by a 35 μlinjection of undiluted antibody. Un-reacted sites were quenched with 35μl of 1M ethanolamine-HCL, pH 8.5 (GE Healthcare, #BR-1000-50). Thisprocedure typically yielded 15,000 response units (RU) of coupledantibody. CTLA-4 ligands human CD80-murine Ig (hCD80-mIg) (Ancell,#510-820) or human CD86-murine Ig (hCD86-mIg) (Ancell, #509-820) werebound to antibody-coated sensor chips by injection of 10 μl or 16 μl,respectively, ligand solution (2 μg/ml protein in HBS-EP buffer [10 mMHEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) surfactant P-20, GEHealthcare, #BR-1001-88]) at a flow rate of 10 μl/min. Ligand capturelevels were typically 135-170 RU. Mutant CTLA-4-Ig proteins (or werediluted in HBS-EP buffer and flowed over ligand-coated sensor chips for2 min at 30 μl/min, followed by 5 min incubation with HBS-EP buffercontaining no protein at the same flow rate. For mutant CTLA-4-Ig fusionproteins having very slow dissociation rates from hCD86-mIg, kineticassays were also conducted using longer dissociation times (e.g., 20min). Rmax signal levels for mutant CTLA-4-Ig proteins ranged fromapproximately 70-100 RU. Regeneration between cycles was performed by 3min incubation with 10 mM glycine buffer (pH 1.7) at 50 μl/min. Newchips were subjected to 4-5 cycles of capture/binding/regeneration priorto use in actual experiments. Data from a reference cell containingrabbit anti-mouse IgG capture antibody alone was subtracted from dataobtained from flow cells containing captured hCD80-mIg or hCD86-mIg.Typically, 8 dilutions of mutant CTLA-4-Ig proteins ranging from 500 nMto 0.2 nM were analyzed against a blank reference (HBS-EP buffer alone).

Sensorgram traces from a typical Biacore analysis are shown in FIG. 5.This figure shows the response (RU) over time (in seconds (s)) generatedby the binding of the following three fusion proteins to the hCD86-mIgfusion protein: (1) Orencia® fusion protein (serving as a control andfor comparison) (Bristol-Myers Squibb Co.; see, e.g., Larson C. P. etal., Am. J. Trans. 5:443-453 (2005)); (2) LEA29Y-Ig fusion protein (forcomparison); and (3) an exemplary mutant CTLA-4-Ig fusion protein of theinvention designated “clone D3” (also termed D3-IgG2). The D3-IgG2fusion protein comprises two identical monomeric fusion proteins thatare covalently linked together by one or more disulfide bonds formedbetween cysteine residues in each monomer. See the discussion in the“SDS/PAGE Analyses” section above. Each monomeric fusion proteincomprises a mature mutant CTLA-4 ECD polypeptide shown in SEQ ID NO:159covalently fused at its C-terminus to the N-terminus of the human IgG2polypeptide shown in EQ ID NO:184 or 218. Other dimeric fusion proteinsof the invention may comprise structures similar to that of the dimericD3-IgG2 fusion protein—except that the D3 mutant CTLA-4 ECD polypeptideof each monomeric fusion protein is replaced by a different mutantCTLA-4 ECD polypeptide.

The association phase reflects the binding between the analyte ofinterest and the ligand of interest. In FIG. 5, the association phasefor each analyte is represented by the curve at times prior to the timemarked by the arrow and is characterized by the binding of analyte(D3-IgG2, Orencia® fusion protein, or LEA29Y-Ig) to the hCD86-mIgligand. The rate at which an analyte associates with the hCD86-mIgligand is reflected in the curve—see, e.g., the sharp rate of increasein the response units beginning at about 510 seconds.

The dissociation phase of the analysis begins at the time marked by thearrow in FIG. 5. During the dissociation phase, the analyte and liganddissociate from their bound conformation. In FIG. 5, the rate at whichan analyte dissociates from the hCD86-mIg ligand is represented by thedecrease in response units (rate of decrease of the response units overtime). Based on these data, the relative dissociation rate constant(“off” rates, k_(off) or k_(d)) and association rate constants (“on”rates, k_(on) or k_(a)) can be determined. The total avidity of theinteraction can be described by the K_(D), (k_(off))/(k_(on)). Increasedbinding avidities are often manifested in slower dissociation rates. Ifthe slower dissociation rate is accompanied by an equal, higher, ormarginally slower association rate, such that the calculated equilibriumdissociation constant K_(D) is lower, the avidity will be greater. Inthis case, a mutant CTLA-4-Ig fusion protein that has a binding avidityfor the hCD86-mIg ligand that is greater than the binding avidity of theOrencia® fusion protein for the same ligand will also have a slowerdissociation rate from the ligand than the Orencia® protein. A mutantCTLA-4-Ig fusion protein that has a binding avidity for the hCD86-mIgligand that is greater than the binding avidity of LEA29Y-Ig for thesame ligand will also have a slower dissociation rate from the ligandthan LEA29Y-Ig.

FIG. 5 shows that the dissociation rate of LEA29Y-Ig from the hCD86-mIgligand is significantly slower than the dissociation rate of theOrencia® protein from the same ligand. LEA29Y-Ig also has an associationrate for binding to hCD86-mIg similar to that observed for the Orencia®fusion protein. Thus, LEA29Y-Ig has a higher avidity for hCD86-mIg thandoes the Orencia® protein. This finding is consistent with previousstudies describing LEA29Y-Ig as having a higher binding avidity for thehCD86-mIg ligand than the Orencia® fusion protein (Larson C. P. et al.,Am. J. Trans. 5:443-453 (2005)). FIG. 5 also shows that the mutantD3-IgG2 fusion protein of the invention has a slower dissociation ratefrom the hCD86-mIg ligand than either the Orencia® or LEA29Y-Ig fusionprotein. The D3-IgG2 fusion protein also has a similar (but somewhatfaster) association rate for binding to hCD86-mIg ligand compared to theassociation rates of either the Orencia® or LEA29Y-Ig fusion proteinsfor hCD86-mIg ligand. Thus, the D3-IgG2 fusion protein exhibits a higherbinding avidity for the hCD86-mIg ligand than either the Orencia® orLEA29Y-Ig fusion protein. Therefore, the D3-IgG2 fusion protein isexpected to bind to native CD86, including, e.g., CD86 as expressed invivo on APCs in mammals, such as humans, with higher binding avidity.This view is further supported by the functional cell-based assaysdiscussed in the Examples below.

Biacore analyses were performed using other mutant CTLA-4-Ig fusionproteins of the invention. In addition, Biacore analyses were performedusing hCD80-mIg-coated sensor chips and mutant CTLA-4-Ig fusion proteinsof the invention. The dissociation rates and binding avidities of thesemutant molecules were determined and compared to the dissociation ratesand binding avidities of the Orencia® and LEA29Y-Ig fusion proteins.Representative results are shown in Tables 3 and 4 and discussed ingreater detail below.

Standard Biacore Data Analyses.

After deletion of the regeneration and capture portions of thesensorgrams, curves were zeroed to a 5 s average of all curvesapproximately 10 s prior to sample injection. A blank curve wassubtracted from each test curve. Data were analyzed by BIAevaluationsoftware (v4.1, available from GE Healthcare) using the “Fit kinetics,Simultaneous k_(a)/k_(d)” function. The injection start time was definedas a time prior to the association phase where all curves were close tozero. Data selection for the association phase began approximately 10 safter the injection start time and ended approximately 10 s prior to theinjection stop time. The injection stop time was defined as a time priorto the appearance of any signal spikes associated with the dissociationphase. The dissociation phase was selected to start 10 s after theinjection stop time and included 280-295 s of the 5 min dissociationphase. The 1:1 Langmuir model describes the reaction A+B<=>AB. Thismodel represents a single ligand binding to a single protein of interest(e.g., receptor). The 1:1 Langmuir model from the BIAevaluation softwarewas used to determine the association rate constant (k_(a)) and thedissociation rate constant (k_(d)) and to calculate the equilibriumdissociation constant, K_(D). K_(D)=k_(d)/k_(a). K_(D)=([A]·[B])/[AB].The equilibrium dissociation constant, K_(D), is equal to the inverse ofthe equilibrium association constant, K_(A). K_(D)=1/K_(A). The rateequations for the reaction (analyte A plus ligand B yielding complexAB), where A=analyte injected, B=free ligand, and t=time, are:d[B]/dt=−(k_(a)[A][B]−k_(d)[AB]) and d[AB]/dt=k_(a)[A][B]−k_(d)[AB].Substituting R, Biacore response units (RU) at a given time, for [AB],Rmax−R for [B], and C (analyte concentration) for [A], the net rateexpression in Biacore units is dR/dt=k_(a)C(Rmax−R)−k_(d)R, where R att0=0, B[0]=Rmax, and AB[0]=0 RU, with the total response=[AB]+RI. Thebulk shift (RI) was set to zero, and Rmax, k_(a) and k_(d) were fitglobally for all curves.

The standard kinetic assays and data analyses described above wereperformed on protein preparations of mutant CTLA-4-Ig fusion protein ofthe invention, as well as LEA29Y-Ig and Orencia® fusion proteins. Tables3 and 4 summarize the binding data for representative mutant CTLA-4-Igfusion proteins of the invention.

Table 3 presents binding avidities of representative mutant CTLA-4-Igfusion proteins to hCD86-mIg, as measured by the standard Biacore assaydescribed above. Specifically, Table 3 shows the name of each mutantCTLA-4Ig fusion protein; the sequence identification number (SEQ ID NO)corresponding to the polypeptide sequence of the monomeric mutant CTLA-4ECD fusion protein; the equilibrium dissociation constant (K_(D) (Molar(M)) determined based on the binding avidity of the mutant protein tothe hCD86-mIg; and the binding avidity of each mutant CTLA-4-Ig to thehCD86-mIg relative to the binding avidity of the Orencia® fusion proteinto the hCD86-mIg. This relative binding avidity (shown in the far-rightcolumn) is shown as a fold improvement in binding avidity of the mutantfusion protein to the hCD86-mIg compared to the binding avidity of theOrencia® fusion protein to the hCD86-mIg. Each mutant CTLA-4-Ig fusionprotein of the invention in Table 3 typically exists in solution as adimeric fusion protein comprising two identical monomeric fusionproteins, wherein each monomeric protein comprises a mutant CTLA-4 ECDpolypeptide (e.g., named D1, D1T, D2, D3, D4, etc.) fused directly atits C-terminus to the N-terminus of an IgG2 Fc polypeptide comprisingthe sequence of SEQ ID NO:184 or 218. Each such dimeric mutant CTLA-4-Igcan be made by using standard techniques known in the art.Alternatively, each such dimeric mutant CTLA-4-Ig fusion protein dimercan be made by using methods set forth in Example 3 above. Briefly, anucleic acid sequence encoding a particular mutant CTLA-4 ECD identifiedin Table 3 (e.g., a nucleic acid sequence encoding a mutant CTLA-4 ECDpolypeptide sequence identified in Table 3) can be cloned into the IgG2Fc fusion vector, mammalian cells can be transfected with the vector,and the resultant fusion protein can be expressed, purified, andevaluated as described in Example 3. An exemplary nucleic acid sequencefor each mutant CTLA-4 ECD is presented in the Sequence Listing includedherewith. The Orencia® fusion protein, which comprises two monomericfusion proteins, each monomeric fusion protein comprising a wild-typehuman CTLA-4 ECD fused to a modified IgG1 Fc, serves as the reference,i.e., with the binding avidity to hCD86-mIg set to 1. The K_(D) valuesfor LEA29Y-Ig fusion protein and the human CTLA-4-Ig fusion protein arealso shown. In addition, the fold improvement in hCD86-mIg bindingavidity of the LEA29Y-Ig compared to the hCD86-mIg binding avidity ofthe Orencia® protein is also shown. The binding avidities of theCTLA-4-IgG2 and Orencia® fusion proteins for hCD86-mIg are approximatelyequal, confirming that the differences between the human IgG2polypeptide present in human CTLA-4-IgG2 and the modified IgG1 presentin the Orencia® protein contribute little, if at all, to the respectivehCD86-mIg binding affinities of these molecules. As is discussed ingreater detail in Example 5 below, we have confirmed that thedifferences in immunosuppressive functional activities between mutantCTLA-4-Ig polypeptides of the invention and the Orencia® protein (orLEA29Y-Ig) cannot be attributed to their respective Ig Fc regionscomprising different IgG isotypes.

As shown in Table 3, representative mutant CTLA-4-Ig fusion proteins ofthe invention have hCD86-mIg binding avidities that are: (1) at leastabout equal to or greater than the binding avidity of human CTLA-4-IgG2(“hCTLA-4-IgG2”) (which comprises two covalently linked monomeric fusionproteins, each such monomeric protein comprising the human CTLA-4 ECDpolypeptide fused to an IgG2 Fc polypeptide); (2) at least about equalto or greater than the binding avidity of the Orencia® protein to thehCD86-mIg ligand; and/or (3) at least about equal to or greater than thebinding avidity of the LEA29Y-Ig to the hCD86-mIg ligand. The foldimprovement in binding avidity to the hCD86-mIg ligand relative to thebinding avidity of the Orencia® protein to hCD86-mIg is indicated foreach CTLA-4-Ig mutant (4^(th) column in Table 3).

A majority of mutant CTLA-4-Ig fusion proteins were found to have a rateof dissociation from hCD86-mIg fusion protein that is slower than therate of dissociation of the Orencia® fusion protein from hCD86-mIg (datanot shown). A number of the mutant CTLA-4-Ig fusion proteins were foundto have a rate of association to hCD86-mIg greater than the rate ofassociation of Orencia® fusion protein to the same ligand (data notshown).

All of the mutant CTLA-4-Ig fusion proteins shown in Table 3 hadhCD86-mIg equilibrium dissociation constants (K_(D)) that were lowerthan the hCD86-mIg equilibrium dissociation constant of humanCTLA-4-IgG2 or Orencia® fusion protein. Furthermore, most of the mutantCTLA-4-Ig fusion proteins shown in Table 3 had hCD86-mIg equilibriumdissociation constants that were lower than hCD86-mIg equilibriumconstant of LEA29Y-Ig.

All of the mutant CTLA-4-Ig fusion proteins shown in Table 3 had bindingavidities for hCD86-mIg that are greater than the binding avidities ofthe human CTLA-4-IgG2 or Orencia® fusion protein for hCD86-mIg (thecalculated fold improvement in hCD86-mIg binding avidity relative to theOrencia® fusion protein is shown the 4^(th) column of Table 3).Moreover, many of the mutant CTLA-4-Ig fusion proteins shown in Table 3had binding avidities for hCD86-mIg that are greater than the bindingavidity of LEA29Y-Ig for hCD86-mIg (4^(th) column of Table 3).

A mutant CTLA-4-Ig of the invention that has a higher binding avidity tothe hCD86-mIg ligand than does the Orencia® or LEA29Y-Ig fusion proteinwill likely have an increased in vivo immunosuppressive potency comparedto the hCTLA-4-IgG2, Orencia®, or LEA29Y-Ig fusion protein,respectively, such as in, e.g., therapeutic and/or prophylactic methodsfor suppressing an immune response in a subject (e.g., in the in vivotreatment of immune system diseases or disorders of mammals, such ase.g., humans, in which immunoinhibition or immunosuppression isdesirable), methods for inhibiting rejection of a tissue or organtransplant from a donor by a recipient (e.g., by a mammal, such as,e.g., a human), and other methods described elsewhere herein.

TABLE 3 SEQ ID NO* Binding Avidity (e.g., of for hCD86-mIg Fusion mutant(Relative to Protein CTLA-4 hCD86-mIg K_(D) Orencia ® fusion (dimer)ECD) (M) protein dimer) Human 162  3.95 × 10⁻⁹ 1.32x  CTLA- 4-IgG2Orencia ® 164  5.23 × 10⁻⁹ 1 fusion protein dimer LEA29Y-Ig 166 1.05 ×10⁻⁹-5.23 × 10⁻¹⁰  5x-10x D1-IgG2 58 2.65 × 10⁻⁹-5.23 × 10⁻¹⁰  2x-10xD1T-IgG2 59 5.23 × 10⁻¹⁰-2.62 × 10⁻¹⁰ 10x-20x D2-IgG2 60 2.62 ×10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40x D3-IgG2 61 <1.31 × 10⁻¹⁰ >40x D4-IgG2 62 2.65× 10⁻⁹-5.23 × 10⁻¹⁰  2x-10x D5-IgG2 63 2.65 × 10⁻⁹-5.23 × 10⁻¹⁰  2x-10xD6-IgG2 64 2.65 × 10⁻⁹-5.23 × 10⁻¹⁰  2x-10x D20-IgG2 65 2.62 ×10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40x D21-IgG2 66 2.62 × 10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40xD23-IgG2 67 <1.31 × 10⁻¹⁰ >40x D24-IgG2 68 <1.31 × 10⁻¹⁰ >40x D26-IgG269 <1.31 × 10⁻¹⁰ >40x D27-IgG2 70 <1.31 × 10⁻¹⁰ >40x D28-IgG2 71 <1.31 ×10⁻¹⁰ >40x D29-IgG2 72 <1.31 × 10⁻¹⁰ >40x D31-IgG2 73 2.62 × 10⁻¹⁰-1.31× 10⁻¹⁰ 20x-40x D3-1-IgG2 1 <1.31 × 10⁻¹⁰ >40x D3-2-IgG2 2 2.62 ×10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40x D3-3-IgG2 3 2.62 × 10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40xD3-4-IgG2 4 <1.31 × 10⁻¹⁰ >40x D3-5-IgG2 5 2.62 × 10⁻¹⁰-1.31 × 10⁻¹⁰20x-40x D3-6-IgG2 6 2.62 × 10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40x D3-7-IgG2 7 <1.31× 10⁻¹⁰ >40x D3-8-IgG2 8 2.62 × 10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40x D3-9-IgG2 9<1.31 × 10⁻¹⁰ >40x D3-11-IgG2 10 <1.31 × 10⁻¹⁰ >40x D3-12-IgG2 11 <1.31× 10⁻¹⁰ >40x D3-14-IgG2 12 <1.31 × 10⁻¹⁰ >40x D3-15-IgG2 13 <1.31 ×10⁻¹⁰ >40x D3-16-IgG2 14 <1.31 × 10⁻¹⁰ >40x D3-17-IgG2 15 <1.31 ×10⁻¹⁰ >40x D3-19-IgG2 16 2.62 × 10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40x D3-20-IgG2 172.62 × 10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40x D3-21-IgG2 18 2.62 × 10⁻¹⁰-1.31 ×10⁻¹⁰ 20x-40x D3-22-IgG2 19 2.62 × 10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40x D3-23-IgG220 2.62 × 10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40x D3-24-IgG2 21 2.62 × 10⁻¹⁰-1.31 ×10⁻¹⁰ 20x-40x D3-25-IgG2 22 2.62 × 10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40x D3-26-IgG223 2.62 × 10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40x D3-27-IgG2 24 2.62 × 10⁻¹⁰-1.31 ×10⁻¹⁰ 20x-40x D3-28-IgG2 25 5.23 × 10⁻¹⁰-2.62 × 10⁻¹⁰ 10x-20x D3-29-IgG226 <1.31 × 10⁻¹⁰ >40x D3-30-IgG2 27 2.62 × 10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40xD3-31-IgG2 28 <1.31 × 10⁻¹⁰ >40x D3-32-IgG2 29 <1.31 × 10⁻¹⁰ >40xD3-33-IgG2 30 <1.31 × 10⁻¹⁰ >40x D3-34-IgG2 31 2.62 × 10⁻¹⁰-1.31 × 10⁻¹⁰20x-40x D3-39-IgG2 32 5.23 × 10⁻¹⁰-2.62 × 10⁻¹⁰ 10x-20x D3-50-IgG2 332.62 × 10⁻¹⁰-1.31 × 10⁻¹⁰ 20x-40x D3-52-IgG2 34 5.23 × 10⁻¹⁰-2.62 ×10⁻¹⁰ 10x-20x D3-53-IgG2 35 <1.31 × 10⁻¹⁰ >40x D3-54-IgG2 36 <1.31 ×10⁻¹⁰ >40x D3-56-IgG2 38 <1.31 × 10⁻¹⁰ >40x D3-62-IgG2 44 <1.31 ×10⁻¹⁰ >40x D3-65-IgG2 47 <1.31 × 10⁻¹⁰ >40x D3-66-IgG2 48 <1.31 ×10⁻¹⁰ >40x D3-69-IgG2 50 <1.31 × 10⁻¹⁰ >40x D3-70-IgG2 51 <1.31 ×10⁻¹⁰ >40x D3-71-IgG2 52 <1.31 × 10⁻¹⁰ >40x D3-72-IgG2 53 <1.31 ×10⁻¹⁰ >40x D3-73-IgG2 54 <1.31 × 10⁻¹⁰ >40x D3-74-IgG2 55 <1.31 ×10⁻¹⁰ >40x D3-75-IgG2 56 <1.31 × 10⁻¹⁰ >40x D3-76-IgG2 57 <1.31 ×10⁻¹⁰ >40x *Note: The sequence identification numbers (SEQ ID NOS) shownin Table 3 for human CTLA-4-IgG2, Orencia ®, and LEA29Y-Ig fusionproteins are those of the polypeptide sequences of these Ig fusionproteins, respectively. The SEQ ID NO shown in Table 3 for each mutantCTLA-4-Ig fusion protein identifies the polypeptide sequence of themutant CTLA-4 ECD of the identified mutant CTLA-4-Ig fusion protein. Thedata pertain to a mutant CTLA-4-Ig comprising a mutant CTLA-4 ECD (e.g.,any of SEQ ID NOS: 1-48 and 58-73) fused at its N-terminus to theC-terminus of the IgG2 Fc polypeptide shown in SEQ ID NO: 184 or 218.Methods for making such fusion proteins are known in the art and aredescribed herein. However, as noted above, we have found experimentallythat a mutant CTLA-4-Ig made in CHO cells using a vector comprising anucleotide sequence encoding the predicted hIgG2 Fc polypeptide shown inSEQ ID NO: 184 does not typically include the predicted C-terminallysine (K) residue; thus, the hgG2 Fc polypeptide sequence of a mutantCTLA-4-IgG2 is that shown in SEQ ID NO: 218, which hIgG2 Fc polypeptidesequence does not include the C-terminal lysine residue as compared tothe sequence shown in SEQ ID NO: 184. Exemplary fusion protein sequenceswhich do not include the C-terminal lysine residue are shown in SEQ IDNOS: 205-214, 219, and 221.

Table 4 presents binding avidities of mutant CTLA-4-Ig fusion proteinsto hCD80-mIg, as measured by the standard Biacore assay. Specifically,Table 4 shows the clone name of a mutant CTLA-4-Ig fusion protein; thesequence identification number (SEQ ID NO) corresponding to thepolypeptide sequence of the monomeric mutant CTLA-4 ECD fusion protein;the equilibrium dissociation constant (K_(D) (Molar (M)) for thehCD80-mIg ligand binding avidity assay; and the binding avidity of themutant CTLA-4-Ig fusion protein to the hCD80-mIg ligand relative to thebinding avidity of the Orencia® fusion protein to the same ligand. Foreach mutant protein, the fold improvement in binding avidity to thehCD80-mIg ligand compared to the binding avidity of the Orencia® fusionprotein to the hCD80-mIg ligand is shown (4^(th) column in Table 4). TheOrencia® fusion protein serves as the reference, i.e., with the bindingavidity to hCD80-mIg set to 1. The mutant CTLA-4-Ig fusion proteinstypically exist as dimeric fusion proteins in solution. As shown inTable 4, dimeric mutant CTLA-4-Ig fusion proteins of the invention havebinding avidities to the hCD80-mIg ligand that are: (1) at least aboutequal to or greater than the binding avidity of human CTLA-4-IgG2; (2)at least about equal to or greater than the binding avidity of theOrencia® fusion protein to the hCD80-mIg ligand; and/or (3) at leastabout equal to or greater than the binding avidity of the LEA29Y-Ig tothe hCD80-mIg ligand. As discussed above, each dimeric mutant CTLA-4-Igdimer can be made by using methods set forth in Example 3 above.

A number of mutant CTLA-4-Ig fusion proteins were found to have a rateof dissociation from hCD80-mIg fusion protein that is about equal to orgreater than the rate of dissociation of the Orencia® fusion proteinfrom the same ligand (data not shown). Some mutant CTLA-4-Ig fusionproteins were found to have a rate of association to hCD80-mIg aboutequal to or greater than the rate of association of Orencia® fusionprotein to the same ligand (data not shown).

Many mutant CTLA-4-Ig fusion proteins shown in Table 4 had hCD80-mIgequilibrium dissociation constants (K_(D)) that were lower than thehCD80-mIg equilibrium dissociation constant of human CTLA-4-IgG2 orOrencia® fusion protein. Furthermore, several mutant CTLA-4-Ig fusionproteins shown in Table 4 had hCD80-mIg equilibrium dissociationconstants that were at least about equal to the hCD80-mIg equilibriumconstant of LEA29Y-Ig.

Many mutant CTLA-4-Ig fusion proteins shown in Table 4 had bindingavidities for hCD80-mIg that are greater than the binding avidities ofhuman CTLA-4-IgG2 or Orencia® fusion protein for the same ligand (foldimprovement in hCD80-mIg binding avidity relative to the Orencia® fusionprotein is shown the 4^(th) column). Additionally, several mutantCTLA-4-Ig fusion proteins shown in Table 4 had binding avidities forhCD80-mIg that are at least about equal to the binding avidity ofLEA29Y-Ig for the same ligand.

A mutant CTLA-4-Ig of the invention that has a higher binding avidity tothe hCD80-mIg than does the hCTLA-4-IgG1, Orencia®, or LEA29Y-Ig fusionprotein will likely have an increased in vivo immunosuppressive potencycompared to the hCTLA-4-IgG2, Orencia®, or LEA29Y-Ig fusion protein,respectively, such as in, e.g., therapeutic and/or prophylactic methodsfor suppressing an immune response in a subject (e.g., in the in vivotreatment of immune system diseases or disorders of mammals, such ase.g., humans, in which immunoinhibition or immunosuppression isdesirable), methods for inhibiting rejection of a tissue or organtransplant from a donor by a recipient (e.g., by a mammal, such as,e.g., a human), and other methods described elsewhere herein.

TABLE 4 SEQ ID NO* Binding Avidity (e.g., of for hCD80-mIg Fusion mutant(Relative to Protein CTLA-4 hCD80-mIg K_(D) Orencia ® fusion (dimer)ECD) (M) protein dimer) hCTLA-4- 162   6.55 × 10⁻¹⁰ 1.34x   IgG2Orencia ® 164   8.77 × 10⁻¹⁰ 1 fusion protein dimer LEA29Y-Ig 166 4.39 ×10⁻¹⁰-2.19 × 10⁻¹⁰   2x-4x D1-IgG2 58 <4.39 × 10⁻¹⁰ >2x D1T-IgG2 59<4.39 × 10⁻¹⁰ >2x D2-IgG2 60 <4.39 × 10⁻¹⁰ >2x D3-IgG2 61 1.75 ×10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D4-IgG2 62 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2xD5-IgG2 63 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D6-IgG2 64 1.75 × 10⁻⁹-4.39× 10⁻¹⁰ 0.5x-2x D20-IgG2 65 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D21-IgG2 661.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D23-IgG2 67 <4.39 × 10⁻¹⁰ >2x D24-IgG268 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D26-IgG2 69 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰0.5x-2x D27-IgG2 70 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D28-IgG2 71 1.75 ×10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D29-IgG2 72 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2xD31-IgG2 73 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-1-IgG2 1 1.75 ×10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-2-IgG2 2 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2xD3-3-IgG2 3 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-4-IgG2 4 1.75 ×10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-5-IgG2 5 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2xD3-6-IgG2 6 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-7-IgG2 7 1.75 ×10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-8-IgG2 8 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2xD3-9-IgG2 9 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-11-IgG2 10 1.75 ×10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-12-IgG2 11 <4.39 × 10⁻¹⁰ >2x D3-14-IgG2 121.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-15-IgG2 13 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰0.5x-2x D3-16-IgG2 14 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-17-IgG2 151.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-19-IgG2 16 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰0.5x-2x D3-20-IgG2 17 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-21-IgG2 181.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-22-IgG2 19 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰0.5x-2x D3-23-IgG2 20 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-24-IgG2 211.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-25-IgG2 22 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰0.5x-2x D3-26-IgG2 23 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-27-IgG2 241.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-28-IgG2 25 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰0.5x-2x D3-29-IgG2 26 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-30-IgG2 271.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-31-IgG2 28 <4.39 × 10⁻¹⁰ >2xD3-32-IgG2 29 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-33-IgG2 30 1.75 ×10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-34-IgG2 31 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2xD3-39-IgG2 32 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-50-IgG2 33 1.75 ×10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-52-IgG2 34 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2xD3-53-IgG2 35 <4.39 × 10⁻¹⁰ >2x D3-54-IgG2 36 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰0.5x-2x D3-56-IgG2 38 1.75 × 10⁻⁹-4.39 × 10⁻¹⁰ 0.5x-2x D3-62-IgG2 44<4.39 × 10⁻¹⁰ >2x D3-65-IgG2 47 <4.39 × 10⁻¹⁰ >2x D3-66-IgG2 48 <4.39 ×10⁻¹⁰ >2x D3-69-IgG2 50 <4.39 × 10⁻¹⁰ >2x D3-70-IgG2 51 <4.39 ×10⁻¹⁰ >2x D3-71-IgG2 52 <4.39 × 10⁻¹⁰ >2x D3-72-IgG2 53 <4.39 ×10⁻¹⁰ >2x D3-73-IgG2 54 <4.39 × 10⁻¹⁰ >2x D3-74-IgG2 55 <4.39 ×10⁻¹⁰ >2x D3-75-IgG2 56 <4.39 × 10⁻¹⁰ >2x D3-76-IgG2 57 1.75 × 10⁻⁹-4.39× 10⁻¹⁰ 0.5x-2x *Note: The SEQ ID NOS shown in Table 4 for humanCTLA-4-IgG2, Orencia ®, and LEA29Y-Ig fusion proteins are those of thepolypeptide sequences of these fusion proteins, respectively. The SEQ IDNO shown in Table 4 for each mutant CTLA-4-Ig fusion protein identifiesthe polypeptide sequence of the mutant CTLA-4 ECD of the identifiedmutant CTLA-4-Ig fusion protein. The data pertain to a mutant CTLA-4-Igcomprising a mutant CTLA-4 ECD (e.g., any of SEQ ID NOS: 1-48 and 58-73)fused at its N-terminus to the C-terminus of the IgG2 Fc polypeptideshown in SEQ ID NO: 184 or 218. However, we have found experimentally byLCMS analysis that a mutant CTLA-4-Ig made in CHO cells using a vectorcomprising a nucleotide sequence encoding the predicted hIgG2 Fcpolypeptide shown in SEQ ID NO: 184 does not typically include thepredicted C-terminal lysine (K) residue; thus, the hIgG2 Fc polypeptidesequence of a mutant CTLA-4-IgG2 is that shown in SEQ ID NO: 218, whichhIgG2 Fc polypeptide sequence does not typically include the C-terminallysine residue as compared to the sequence shown in SEQ ID NO: 184.Exemplary fusion protein sequences that do not include the C-terminallysine residue are shown in SEQ ID NOS: 205-214, 219, and 221.

Monomeric Kinetic Assays.

Further confirmation of binding properties of mutant CTLA-4-Ig fusionproteins was obtained by monovalent kinetic binding assays. These assaysmeasure the binding kinetics of a bivalent test protein (e.g., a dimericmutant CTLA-4-Ig fusion protein) coated to sensor chips and monovalentligand (papain-digested hCD86-mIg) in the mobile phase. Goat anti-humanIgG antibody (Jackson ImmunoResearch, #109-005-098) was coupled to CM5sensor chips according to the manufacturer's protocols, typicallyyielding 15,000 response units (RU). Ligand was captured by incubationof antibody-coated sensor chips with 10 μl of a 2 μg/ml solution ofdimeric mutant CTLA-4-Ig fusion proteins in HBS-EP buffer at a flow rateof 10 μl/min, Ligand capture levels were typically 25-80 RU. MonomerichCD86-mIg ligands (created by papain digestion and protein-A sepharoseadsorption of hCD86-mIg, as described in Hermanson, G. T. BIOCONJUGATETECHNIQUES, Academic Press, 1996) were diluted in HBS-EP buffer andflowed through test protein-coated sensor chips for 2 min at 30 μl/min,followed by 2 min incubation with HBS-EP buffer containing no protein atthe same flow rate. Regeneration between cycles was performed by 3 minincubation with 10 mM glycine buffer (pH 1.7) at 30 μl/min. Typically, 8dilutions of monomeric analyte proteins ranging from 3000 nM to 0.2 nMwere analyzed against a blank reference (HBS-EP buffer alone) induplicate. Rmax signal levels for binding to mutant CTLA-4-Ig proteinsranged from 10-60 RU.

For kinetic analysis, data from monomeric binding assays were selectedas described above except that association data selection began andended 5 s from the injection start and stop times, and dissociation dataselection started 5 s after the injection stop time, and typicallyincluded 1-60 s of the dissociation period. Such data were also analyzedfor steady state equilibrium affinities using the BIAevaluationsoftware. Steady state binding levels for each concentration(Response_(eq) (“Req”) values) were averaged over 5-20 s range near theend of the sample injection using the BIAevaluation “General fit:average” function. Steady state affinity was determined from the Req vs.concentration plot using the BIAevaluation software according to theformula Req=K_(A)×C×Rmax/(K_(A)×C×n+1), where C is the analyteconcentration and n, the steric interference factor, is 1, andK_(D)=1/K_(A). In some cases, substantial non-specific binding,represented by a residual flat R-value trace after dissociation, wasobserved. Such data were corrected by subtracting the residual R-valuesfrom the Req values. The K_(D) was then calculated in GraphPad Prismsoftware (GraphPad Software, Inc.) using the “one site-specific binding”model.

This monomeric binding assay was performed on a subset of representativemutant CTLA-4-Ig proteins and the results are summarized in Table 5.Overall, the degree of improvement in hCD86 ECD binding to mutantCTLA-4-Ig fusion proteins relative to LEA29Y-Ig observed in themonomeric binding assays was similar to that observed for hCD86-mIgbinding to mutant CTLA-4-Ig fusion proteins relative to LEA29Y-Ig in thestandard kinetic assays. Such results support the conclusion that theobserved improvements in binding kinetics for the mutant proteins aredue to real improvements in binding affinities of the mutant proteins(e.g., compared to Orencia® and/or LEA29Y-Ig fusion proteins) and arenot due to potential artifacts caused by higher valency of aggregatedprotein preparations.

TABLE 5 Binding Affinity for Monomeric Fusion SEQ ID NO* Monomeric hCD86ECD Protein (e.g., of mutant hCD86 ECD (Relative to (dimer) CTLA-4 ECD)K_(D) (M) LEA29Y-Ig) LEA29Y-Ig 166   1.68 × 10⁻⁶ 1 D3-IgG2 61 <3.36 ×10⁻⁷ >5x D3-4-IgG2 4 <3.36 × 10⁻⁷ >5x D3-11-IgG2 10 <3.36 × 10⁻⁷ >5xD3-12-IgG2 11 <3.36 × 10⁻⁷ >5x D3-14-IgG2 12 <3.36 × 10⁻⁷ >5x D3-17-IgG215 <3.36 × 10⁻⁷ >5x D3-20-IgG2 17 8.4 × 10⁻⁷-3.36 × 10⁻⁷ 2x-5xD3-27-IgG2 24 8.4 × 10⁻⁷-3.36 × 10⁻⁷ 2x-5x D3-29-IgG2 26 <3.36 ×10⁻⁷ >5x D3-31-IgG2 28 <3.36 × 10⁻⁷ >5x D3-53-IgG2 35 <3.36 × 10⁻⁷ >5x*Note: The SEQ ID NO shown in Table 5 for LEA29Y-Ig is that of thepolypeptide sequence of the LEA29Y-Ig fusion protein. The SEQ ID NOshown in Table 5 for each mutant CTLA-4-Ig fusion protein identifies thepolypeptide sequence of the mutant CTLA-4 ECD present in the identifiedmutant CTLA-4-Ig fusion protein. The data pertain to a mutant CTLA-4-Igcomprising a mutant CTLA-4 ECD (e.g., any of SEQ ID NOS: 4, 10-12, 15,17, 24, 26, 28, 35, and 61) fused at its N-terminus to the C-terminus ofthe IgG2 Fc polypeptide shown in SEQ ID NO: 184 or 218. However, we havefound experimentally by LCMS analysis that a mutant CTLA-4-Ig made inCHO cells using a vector comprising a nucleotide sequence encoding thepredicted hIgG2 Fc polypeptide shown in SEQ ID NO: 184 does nottypically include the predicted C-terminal lysine (K) residue; thus, thehIgG2 Fc polypeptide sequence of a mutant CTLA-4-IgG2 is that shown inSEQ ID NO: 208, which hIgG2 Fc polypeptide sequence does not typicallyinclude the C-terminal lysine residue as compared to the sequence shownin SEQ ID NO: 184. Exemplary fusion protein sequences that do notinclude the C-terminal lysine residue are shown in SEQ ID NOS: 205-214,219, and 221. Exemplary fusion protein sequences that include theC-terminal lysine residue are shown in SEQ ID NOS: 74-79, 197-200, 220,and 222.

Example 5 Measuring Biological Activity of CTLA-4 Mutants using HumanPBMC Proliferation Assays Anti-CD3 Antibody Stimulation

CTLA-4-Ig and particular variants thereof have been shown to be potentinhibitors of T cell proliferation in vitro (see, e.g., Larson et al.,Am. J. Transplant. 5, 443). To measure improved activity of mutantCTLA-4-Ig proteins in such assays, a peripheral blood mononuclear cell(PBMC) proliferation assay was developed.

Human blood (freshly collected from a donor program) was diluted with anequal volume of PBS and fractionated to isolate PBMCs using a Histopaque(Sigma, #10771) Ficoll gradient as per the manufacturer's recommendedconditions. PBMCs were diluted in Growth Medium (DMEM/F12 medium(Invitrogen, #10565-018) supplemented with 10% FBS (Hyclone #SV30014.03)and 1×PSG (Invitrogen, #10378-016) and added to 96-well culture plates(BD Biosciences, #353077) at a density of 1×10⁵ cells/well. Testcompounds were serially diluted in Growth Medium and added to wells intriplicate. Cell proliferation was initiated by addition of mouseanti-human CD3 antibody (BD Pharmingen: 555329) to a final concentrationof 5 μg/ml. After incubation at 37° C. for 2 days (d), ³H thymidine (GEHealthcare, #TRK758-5MCI) was added at 1 μCi/well and plates wereincubated at 37° C. for an additional 16 hr. Cells were harvested with acell harvester (FilterMate Omnifilter-96 Harvester) using themanufacturer's recommended conditions and measured for ³H thymidineincorporation using a scintillation counter (Wallac Trilux, #1450-421).The degree of ³H thymidine incorporation (³H thymidine uptake) isindicative of the degree of T cell proliferation. ³H-thymidineincorporation is measured by standard techniques. Proliferation of Tcells is expressed as the mean counts per minute (cpm) of triplicatewells.

Cell proliferation data was analyzed with GraphPad Prism 5 softwareusing a non-linear regression curve fit model (sigmoidal dose-response,variable slope) and the least squares fit method. The IC50 (also shownas IC₅₀) parameters and their associated 95% confidence intervals fromthe results are reported (Table 6). FIG. 6 shows cell proliferationcurves from a representative PBMC proliferation assay (using anti-CD3antibody stimulation) involving a set of exemplary mutant CTLA-4-Igfusion proteins of the invention—i.e., D3-04-IgG2, D3-11-IgG2,D3-12-IgG2, and D3-14-IgG2 fusion proteins. The graph is a plot of ³Hthymidine incorporation (counts per minute (cpm)) versus proteinconcentration (nanoMolar (nM)). ³H thymidine incorporation (3H thymidineuptake), which is indicative of the degree of cell proliferation, ismeasured by standard techniques.

The Orencia® and LEA29Y-Ig fusion proteins were included as controls forcomparison. These results demonstrate that mutant CTLA-4-Ig Ig fusionproteins of the invention have significantly higher potency or greaterability than the Orencia® and/or LEA29Y-Ig fusion protein(s) ininhibiting or suppressing polyclonal T cell activation or T cellproliferation in vitro.

The PBMC proliferation assay was performed using other mutant CTLA-4-Igfusion proteins of the invention. Table 6 provides a summary of the datafor a representative set of mutant CTLA-4-Ig fusion proteins of theinvention. Table 6 presents comparisons of mean IC50 values (nanomolar(nM)) for exemplary mutant CTLA-4-Ig fusion proteins versus controls(Orencia®, hCTLA-4-IgG2, and LEA29Y-Ig fusion proteins) in the PBMCproliferation assay (with anti-CD3 antibody stimulation). An IC50 valuerepresents the concentration of a compound (e.g., mutant CTLA-4-Ig,hCTLA-4-IgG2, Orencia®, or LEA29Y-Ig fusion protein) that is requiredfor 50% inhibition of T cell proliferation in vitro. IC50 values fromindividual experiments were averaged to provide mean IC50 values, whichwere used for statistical analyses. A one-way ANOVA with post hocDunnett or Bonferroni test was used to compare mutant CLTA-4-Ig fusionproteins and hCTLA-4-IgG2 to the Orencia® or LEA29Y-Ig fusion protein,respectively (C. W. Dunnett, New Tables for Multiple Comparisons with aControl, Biometrics 20(3):482-491 (September 1964); Abdi, Herve, “TheBonferroni and Sidak corrections for multiple comparisons”, inENCYCLOPEDIA OF MEASUREMENT AND STATISTICS (N.J. Salkind ed., ThousandOaks, Calif. 2007); also available on the worldwide web at the webaddress utdallas.edu/˜herve/Abdi-Bonferroni2007-pretty.pdf.).Statistical analysis of Ig fusion protein composed of one of thefollowing mutant CTLA-4 ECD polypeptides—clones D24, D3-07, D3-15 andD3-16—was not performed as n=1. The term “SD (mean log IC50)” representsthe standard deviation in mean log IC50 values.

TABLE 6 Summary of Data From Exemplary PBMC Proliferation Assays UsingAnti-CD3 Antibody Stimulation Fusion Protein Mean IC50 Mean Log IC50 SD(Mean Log (dimer) (nM) (nM) IC50) (nM) Orencia ® fusion 5.16 0.71 0.51protein dimer hCTLA-4-IgG2 8.46 0.93 0.43 LEA29Y-Ig 0.48¹ −0.32 0.54D3-4-IgG2 0.05^(1,2) −1.27 0.45 D3-7-IgG2 0.12 −0.93 NA D3-11-IgG20.08^(1,2) −1.07 0.40 D3-12-IgG2 0.06^(1,2) −1.27 0.17 D3-14-IgG20.07^(1,2) −1.17 0.15 D3-15-IgG2 0.21 −0.69 NA D3-17-IgG2 0.04^(1,2)−1.36 0.37 D3-20-IgG2 0.07^(1,2) −1.19 0.37 D3-26-IgG2 0.15¹ −0.83 0.49D3-27-IgG2 0.10^(1,2) −1.00 0.41 D3-29-IgG2 0.07^(1,2) −1.13 0.53D3-30-IgG2 0.18¹ −0.74 0.35 D3-31-IgG2 0.04^(1,2) −1.42 0.25 D3-32-IgG20.10¹ −1.00 0.47 D3-33-IgG2 0.12¹ −0.94 0.69 D3-34-IgG2 0.41¹ −0.39 0.18D3-39-IgG2 0.47¹ −0.33 0.21 D3-50-IgG2 0.35¹ −0.46 0.23 D3-52-IgG2 0.49¹−0.31 0.20 D3-53-IgG2 0.02^(1,2) −1.69 0.24 D3-54-IgG2 0.17¹ −0.77 0.22D3-56-IgG2 0.08¹ −1.08 0.20 D3-62-IgG2 0.05^(1,2) −1.29 0.23 D3-69-IgG20.05^(1,2) −1.34 0.19 D3-70-IgG2 0.08¹ −1.10 0.10 D3-71-IgG2 0.05 −1.32NA D3-72-IgG2 0.03^(1,2) −1.54 0.19 D3-73-IgG2 0.23¹ −0.64 0.25D3-75-IgG2 0.08¹ −1.11 0.33 D3-76-IgG2 0.13¹ −0.88 0.36 D3-IgG2 0.14¹−0.85 0.13 D24-IgG2 0.12 −0.93 NA The superscripts shown in Table 6 areas follows: ¹Statistically different to Orencia with p < 0.05, asdetermined by 1 way ANOVA with post hoc Dunnett test. ²Statisticallydifferent to LEA with p < 0.05, as determined by 1 way ANOVA post hocBonferroni test. The fusion proteins comprising D3-7, D3-15, D3-71 andD24 mutant CTLA-4 ECDs were tested once and therefore no statisticalcomparison could be performed. Note: The term “NA” in Table 6 means “notavailable”.

Statistical analysis revealed that all of the mutant CTLA-4-Ig fusionproteins that were tested in at least two separate assays and aredesignated with the superscript (1) (see Table 6) were statisticallysuperior in potency to the Orencia® and hCTLA-4-IgG2 fusion proteins(p<0.05) (i.e., have a greater ability to suppress or inhibit T cellproliferation in in vitro PBMC assays than the Orencia® and hCTLA-4-IgG2fusion proteins). Those designated with the superscript (2) (see Table6) were also statistically superior in potency to the LEA29Y-Ig fusionprotein (p<0.05) (i.e., have a greater ability to suppress or inhibit Tcell proliferation in in vitro PBMC assays than the LEA29Y-Ig fusionprotein), as determined by one-way ANOVA with post hoc Dunnett andBonferroni tests discussed supra. There was no statistical differencebetween human CTLA-4 fusion proteins comprising either the modified IgG1Fc (as in Orencia®) or the wild-type IgG2 Fc (as in hCTLA4-IgG2). Thisfinding implies that the differences in functional activities shown inTable 6 between the mutant CTLA-4-Ig fusion proteins of the invention(which each comprise a human IgG2 Fc) and either Orencia® or hCTLA4-IgG2were as a direct consequence of amino acid changes (i.e., amino acidsubstitutions) made in the CTLA-4 ECD region. The differences infunctional activities between these mutant CTLA-4-Ig fusion proteins ofthe invention and, for example, Orencia® (which comprises a modifiedIgG1 Fc) were not due to differences in their respective Ig Fcpolypeptide sequences.

It is believed that given the increased abilities of mutant CTLA-4-Igfusion proteins of the invention to suppress or inhibit T cellproliferation in in vitro assays as compared to Orencia®, LEA29Y-Ig,and/or hCTLA-4-IgG2 fusion proteins, such mutant proteins should alsoexhibit increased immunosuppressive potencies in in vivo therapeuticand/prophylactic methods as compared to Orencia®, LEA29Y-Ig, and/orhCTLA-4-IgG2 fusion proteins. Each such mutant CTLA-4-Ig fusion proteinof the invention is believed to have a greater ability to suppress orinhibit T cell proliferation in in vivo methods or applications relativeto Orencia®, LEA29Y-Ig, and/or hCTLA-4-IgG2 fusion proteins, such as in,e.g., therapeutic and/or prophylactic methods for suppressing orinhibiting an immune response in a subject (e.g., in the in vivotreatment of immune system diseases or disorders in a mammal, such ase.g., a human), methods for suppressing or inhibiting rejection of atissue or organ transplant from a donor by a recipient (e.g., by amammal, such as e.g., a human), and/or other treatment or diagnosticmethods described elsewhere herein.

Applying the same statistical analyses, we found no statisticaldifference between the Orencia® fusion protein (comprising human CTLA-4ECD-mutant IgG1) and human CTLA-4-IgG2. Thus, it is believed that thedifferences in the Ig domains of these molecules (i.e., the mutant IgG1of the Orencia® fusion protein and the human IgG2 of hCTLA-4-IgG2) didnot affect the functionality of these molecules. See FIG. 11. Theinhibition of proliferation observed with increasing doses of theOrencia® fusion protein was not significantly different from thatobserved with CTLA-4-IgG2, indicating that their respectiveimmunosuppressive activities are not biased according to their differentIgG isotypes, but rather result from their hCTLA-4 ECD polypeptides.Thus, the different immunosuppressive activities between mutantCTLA-4-Ig polypeptides of the invention and Orencia® fusion protein (orLEA29Y-Ig, since it contains the same Ig as in the Orencia® protein)cannot be attributed to their respective Fc regions comprising differentIgG isotypes.

The invention includes monomeric mutant CTLA-4 ECD proteins that have anability and, in some instances, a greater ability to suppress or inhibitT cell activation or proliferation than a monomeric human CTLA-4 proteinor an extracellular domain thereof. Also provided are monomeric mutantCTLA-4 ECD fusion proteins that have an ability and, in some instances,a greater ability to suppress or inhibit T cell activation orproliferation than a monomeric hCTLA-4 Ig fusion protein or anextracellular domain thereof. Also included are mutant CTLA-4 ECDprotein dimers that have an ability and, in some instances, a greaterability to suppress or inhibit T cell activation or proliferation than adimer comprising two human CTLA-4 extracellular domains. Some mutantCTLA-4 ECD fusion protein dimers of the invention (e.g., mutantCTLA-4-ECD-Ig fusion protein dimers) have an ability and, in someinstances, a greater ability to suppress or inhibit T cell activation orproliferation than a hCTLA-4-IgG2 fusion protein dimer, Orencia® fusionprotein dimer, and/or LEA29YIg fusion protein dimer.

Example 6 Measuring Biological Activity of Mutant CTLA-4-Ig MoleculesUsing Human CD4⁺ T Cell Proliferation Assays

Human CTLA-4-Ig and particular variants thereof have been shown toinhibit T cell proliferation by blocking signaling of CD80 and CD86though CD28 (Linsley P. S., Immunity 1:793-801 (1994); Larson C. P. etal., Am. J. Transplant. 5:443-453 (2005)). Because the mutant CTLA-4-Igproteins of the invention are improved in binding avidity for theCD86-Ig ligand, a CD4⁺ T cell proliferation assay was developed tomeasure activity of the mutant CTLA-4-Ig proteins in blocking signalingthrough CD86.

Creation of DNA Sequence Encoding a Full-length Human CD86 Protein.

Plasmid pcDNA3.1 hB7.2 FL was created to encode the full-length humanCD86 protein for expression on the surface of transfected cells. DNAencoding human CD86 was generated by PCR amplification of cDNA derivedfrom human leukocytes (BD Biosciences, Cat#HL4050AH) using forward andreverse oligonucleotide primers designed based on sequence homology tothe CD86-encoding nucleotide sequence set forth in SEQ ID NO:176. Theprimers were designed, made, and assembled using standard techniqueswell known by those of ordinary skill in the art and included stop andstart codons and restriction sites as necessary. The PCR amplificationprocedures employed are also well known in the art. Such techniques aredescribed in, e.g., Berger, Ausubel, and Sambrook, all supra. 50nanogram (ng) cDNA was used as template in a 100 μl PCR reaction with 1μM forward and reverse primers, Herculase Polymerase buffer (Stratagene;#600260) and 200 μM dNTPs for 30 amplification cycles (94° C., 30 s; 50°C., 30 s; 72° C., 60 s). The PCR product was purified by QiaQuick PCRSpin Columns (Qiagen #28106) and digested with restriction enzymes KpnIand NotI. Fragments were separated by agarose-gel electrophoresis,purified using Qiaquick Gel Extraction Kit (Qiagen, #28704) as permanufacturer's recommendation, and ligated into similarly digestedplasmid pcDNA 3.1(+) (Invitrogen, Cat. #V790-20). Ligations weretransformed into TOP10 E. coli cells (Qiagen, Cat. #C4040-10) as permanufacturer's recommendations. Transformed cells were incubated in LB(Luria broth media) containing 50 μg/ml carbenicillin at 250 rpmovernight at 37° C. and then used to make a maxiprep (Qiagen; #12362)stock of plasmid DNA as per the manufacturer's recommended conditions.

The predicted amino acid sequence of the full-length human CD86 proteinis shown in SEQ ID NO:175. In this sequence, amino acid residues 1-23comprise the predicted signal sequence, amino acid residues 24-241comprise the human CD86 extracellular domain, amino acid residues242-270 comprise the transmembrane domain, and amino acid residues271-329 comprise the cytoplasmic domain.

Creation of Stable Cell Lines Expressing Human CD86 on the Cell Surface.

HEK293 cells were grown to 80-90% confluence in T-75 flasks containing20 ml Growth Medium (DMEM/F12 medium (Invitrogen, Cat. #10565-018)supplemented with 10% FBS (Hyclone Cat. #SV30014.03) and 1×PSG(Invitrogen, Cat. #10378-016)). Cells were transfected with 10 μgplasmid DNA (pcDNA3.1 hB7.2 FL) mixed with 60 μl Fugene 6 (ROCHE,#11814443001) as per the manufacturer's recommended conditions. Cellswere incubated for 2 days (d) at 37° C. in Growth Medium and furtherincubated for 10 d at 37° C. in Selection Medium (Growth Mediumcontaining 300 μg/ml Geneticin (Invitrogen, #10131-027), changing themedia every 2 d. To enable FACS sorting, transfected cells were stainedwith FITC-labeled anti-CD86 antibody (BD Biosciences, #555) as per themanufacturer's recommend conditions. Using a cell-sorter (Dako, MoFlo)gated for FITC signal, CD86-positive cells were individually sorted into96-well culture plates (Sigma-Aldrich, #CLS-3596) containing 200 μl/wellGrowth Medium containing 25% Conditioned Medium (Growth Mediumpreviously harvested from untransfected (or naïve) cell cultures). Afterincubation at 37° C. for 13-19 d, cells were dispersed by trypsinhydrolysis and transferred to 24-well culture plates containing 0.5ml/well of Growth Medium. After incubation at 37° C. for 7 d, cells weredispersed by trypsin hydrolysis and transferred to T-75 flaskscontaining 20 ml Growth Medium. Final cell lines were selected basedupon high levels of cell-surface CD86 expression as measured by FACSanalysis of FITC-labeled anti-CD86 antibody stained cells using FACSCaliber (BD Biosciences), as per the manufacturer's recommendedconditions.

CD4⁺ T cell Proliferation Assays.

CD4⁺ T cells were enriched to >96% from human Buffy coat preps (StanfordUniversity Blood Center, Stanford, Calif.) using the EasySep Human CD4Positive Selection Kit (StemCell Technologies, #18052R) with a magneticcell separator (RoboSep, StemCell Technologies, #20000) followingmanufacturer's recommendations. Enriched CD4⁺ T cells were adjusted to adensity of 1×10⁶ cells/ml in Yssel's media (Gemini Bio-Products,#400-102) supplemented with 10% FBS (Hyclone SV30014.03) and added to 96well tissue culture plates at 50 μl/well. HEK293 cells expressingmembrane-bound human CD86 were irradiated at 6000 rads (StanfordResearch Institute, Menlo Park, Calif.), adjusted to 1×10⁶ cells/ml inthe same medium and added to the culture plates at 50 μl/well. Testcompounds were serially diluted in the same medium and added to wells intriplicate. Cell proliferation was initiated by addition of mouseanti-human CD3 antibody (BD Pharmingen: 555329) to a final concentrationof 5 μg/ml. After incubation at 37° C. for 3 d, ³H thymidine (GEHealthcare, #TRK758-5MCI) was added at 1 μCi/well and plates wereincubated at 37° C. for an additional 18 hr. Cells were harvested usinga cell harvester (Perkin Elmer Filter Harvester D961962) and ³Hthymidine was measured using a liquid scintillation counter (WallacTrilux 1450) as per the manufacturer's recommended conditions. Cellproliferation data were analyzed with GraphPad Prism 5 software using avariable slope equation(Y=Bottom+(Top−Bottom)/(1+10̂((LogIC50−X)(HillSlope))) to generate anIC50 for each test compound. The term “(LogIC50−X)(HillSlope)” is anexponent in the equation.

FIG. 7 shows cell proliferation curves from representative CD4⁺ T cellproliferation assays involving an exemplary set of mutant CTLA-4-Igfusion proteins of the invention—i.e., D3-04-IgG2, D3-11-IgG2,D3-12-IgG2, and D3-14-IgG2. Orencia® and LEA29Y-Ig fusion proteins wereincluded as controls for comparison. The graph is a plot of ³H thymidineincorporation (cpm) versus concentration (nM) of protein. ³H thymidineincorporation is indicative of the degree of cell proliferation and ismeasured by standard techniques. These results demonstrate that mutantCTLA-4-Ig fusion proteins of the invention have significantly higherpotency or greater ability than the Orencia® and/or LEA29Y-Ig fusionproteins in inhibiting or suppressing CD86 co-stimulation in vitro.

This CD4⁺ T cell proliferation assay was performed on a number of othermutant CTLA-4-Ig fusion proteins of the invention. Table 7 provides asummary of the data for a set of exemplary mutant CTLA-4-Ig fusionproteins of the invention. Table 7 presents comparisons of mean IC50values (nanomolar (nM)) for exemplary mutant CTLA-4-Ig fusion proteinsversus reference controls (Orencia®, LEA29Y-Ig, and human CTLA-4-IgG2fusion proteins) using the CD4⁺ T cell proliferation assay. IC50s fromindividual experiments were averaged to provide mean IC50 values, whichwere used for statistical analyses. The term “SD (mean log IC50)”represents the standard deviation in mean log IC50 values.

TABLE 7 Summary of Data From Exemplary CD4⁺ T Cell Proliferation AssaysFusion Protein Mean IC50 Mean Log IC50 SD (Mean Log (dimer) (nM) (nM)IC50) (nM) Orencia ® fusion 1.56 0.19 0.22 protein dimer hCTLA-4-IgG22.24 0.35 0.28 LEA29Y-Ig 0.21¹ −0.67 0.25 D3-02-IgG2 0.03 −1.56 NAD3-03-IgG2 0.04^(1,2) −1.40 0.11 D3-04-IgG2 0.03^(1,2) −1.47 0.02D3-06-IgG2 0.05¹ −1.32 0.03 D3-11-IgG2 0.03^(1,2) −1.52 0.07 D3-12-IgG20.04^(1,2) −1.45 0.09 D3-14-IgG2 0.04^(1,2) −1.37 0.11 D3-15-IgG20.02^(1,2) −1.62 0.08 D3-17-IgG2 0.03^(1,2) −1.47 0.11 D3-20-IgG20.04^(1,2) −1.40 0.11 D3-27-IgG2 0.04^(1,2) −1.37 0.10 D3-29-IgG20.04^(1,2) −1.36 0.13 D3-31-IgG2 0.03^(1,2) −1.58 0.21 D3-34-IgG20.05^(1,2) −1.28 0.13 D3-39-IgG2 0.05^(1,2) −1.35 0.10 D3-50-IgG20.06^(1,2) −1.23 0.24 D3-52-IgG2 0.07^(1,2) −1.14 0.24 D3-53-IgG20.02^(1,2) −1.70 0.25 D3-54-IgG2 0.04^(1,2) −1.40 0.12 D3-56-IgG20.04^(1,2) −1.41 0.07 D3-62-IgG2 0.04^(1,2) −1.44 0.09 D3-65-IgG20.06^(1,2) −1.26 0.14 D3-69-IgG2 0.03^(1,2) −1.59 0.14 D3-70-IgG20.05^(1,2) −1.30 0.06 D3-71-IgG2 0.04^(1,2) −1.43 0.09 D3-72-IgG20.03^(1,2) −1.48 0.05 D3-73-IgG2 0.06^(1,2) −1.26 0.18 D3-75-IgG20.03^(1,2) −1.51 0.12 D2-IgG2 0.03^(1,2) −1.52 0.18 D3-IgG2 0.04^(1,2)−1.45 0.17 The superscripts shown in Table 7 are as follows:¹Statistically different to Orencia with p < 0.05, as determined by 1way ANOVA with post hoc Dunnett test. ²Statistically different to LEAwith p < 0.05, as determined by 1 way ANOVA post hoc Bonferroni test.The fusion protein comprising the D3-02 mutant CTLA-4 ECD was testedonce and therefore no statistical comparison could be performed.

Statistical analysis revealed that all of the mutant CTLA-4-Ig fusionproteins that were tested in at least two separate assays and aredesignated with the superscript (1) (see Table 7) were statisticallysuperior in potency to the Orencia® and hCTLA-4-IgG2 fusion proteins(p<0.05) (i.e., have a greater ability to suppress or inhibit T cellproliferation in in vitro CD4⁺ T cell assays than the Orencia® andhCTLA-4-IgG2 fusion proteins). Those designated with the superscript (2)(see Table 7) were also statistically superior in potency to theLEA29Y-Ig fusion protein (p<0.05) (i.e., have a greater ability tosuppress or inhibit T cell proliferation in in vitro CD4⁺ T cell assaysthan the LEA29Y-Ig fusion protein), as determined by one-way ANOVA withpost hoc Dunnett and Bonferroni tests discussed supra. There was nostatistical difference between human CTLA-4 fusion proteins comprisingeither the modified IgG1 Fc (as in Orencia®) or the wild-type IgG2 Fc(as in hCTLA4-IgG2). This finding implies that the differences infunctional activities shown in Table 7 between the mutant CTLA-4-Igfusion proteins of the invention (which each comprise a human IgG2 Fc)and either Orencia® or hCTLA4-IgG2 were as a direct consequence of aminoacid changes (i.e., amino acid substitutions) made in the CTLA-4 ECDregion. The differences in functional activities between these mutantCTLA-4-Ig fusion proteins of the invention and, for example, Orencia®(which comprises a modified IgG1 Fc) were not due to differences intheir respective Ig Fc polypeptide sequences.

It is believed that given the increased abilities of mutant CTLA-4-Igproteins of the invention to suppress or inhibit CD86-mediatedco-stimulation of T cells (e.g., human T cells) in in vitro assays ascompared to Orencia®, LEA29Y-Ig, and/or hCTLA-4-IgG2 fusion proteins,such mutant proteins should also exhibit increased immunosuppressivepotencies in in vivo therapeutic and/prophylactic methods orapplications as compared to Orencia®, LEA29Y-Ig, and/or human CTLA-4-Igfusion proteins (e.g., human CTLA-4-IgG2 (“hCTLA-4-IgG2”)),respectively. In one aspect, mutant CTLA-4-Ig fusion proteins of theinvention are believed to have a greater ability to suppress or inhibitCD86-mediated co-stimulation of T cells (e.g., human T cells) in in vivomethods or applications compared to Orencia®, LEA29Y-Ig, and/or humanCTLA-4-Ig (e.g., hCTLA-4-IgG2) fusion proteins, respectively, such asin, e.g., therapeutic and/or prophylactic methods for suppressing orinhibiting an immune response in a subject (e.g., in the in vivotreatment of immune system diseases or disorders in e.g., a mammal, suchas, e.g., a human), methods for suppressing or inhibiting rejection of atissue or organ transplant from a donor by a recipient (e.g., by amammal, such as, e.g., a human), and/or other treatment or diagnosticmethods described elsewhere herein.

Example 7 Measuring Biological Activity of Mutant CTLA-4-Ig MoleculesUsing Human PBMC Proliferation Assays Recall Antigen Stimulation

The activation of memory T cells is an important aspect of autoimmunity(Rogers N. J., et al. Eur. J. Immunol. 35:2909-2919 (2005)). To measureimmunosuppressive activity of mutant CTLA-4-Ig proteins in this respect,a peripheral blood mononuclear cell (PBMC) proliferation assay wasdeveloped using PPD antigen stimulation.

Human blood (freshly collected from a donor program) was diluted with anequal volume of PBS and fractionated to isolate PBMCs using a Histopaque(Sigma, #10771) Ficoll gradient as per the manufacturer's recommendedconditions. PBMCs were diluted in RPMI medium (Sigma, #R8758)supplemented with 10% FBS (Hyclone #SV30014.03) and 1×PSG (penicillin,streptomycin and glutamine) (Invitrogen, #10378-016) and added to96-well culture plates (BD Biosciences, #353077) at a density of 1×10⁵cells/well. Test compounds were serially diluted in the same medium andadded to wells in quadruplicate. Cell proliferation was initiated byaddition of PPD antigen (purified protein derivative from Mycobacteriumtuberculosis, Mycos, #P-1000-001) to a final concentration of 5 μg/ml.After incubation at 37° C. for 5 d, ³H thymidine (GE Healthcare,#TRK758-5MCI) was added at 1 μCi/well and plates were incubated at 37°C. for an additional 18 hr. Cells were harvested with a cell harvester(FilterMate Omnifilter-96 Harvester, Perkin Elmer) using themanufacturer's recommended conditions and measured for ³H thymidineincorporation using a scintillation counter (Wallac Trilux, #1450-421).Cell proliferation data was analyzed with GraphPad Prism 5 softwareusing a non-linear regression curve fit model (sigmoidal dose-response,variable slope) and the least squares fit method. The IC₅₀ (or “IC50”)parameters and their associated 95% confidence intervals are reported.

FIG. 8 shows cell proliferation curves from representative PBMCproliferation assays involving a set of exemplary mutant CTLA-4-Igfusion proteins of the invention—i.e., D3-IgG2, D3-12-IgG2, D3-17-IgG2,and D3-29-IgG2 fusion proteins. Orencia® and LEA29Y-Ig fusion proteinswere included as controls for comparison. The graph is a plot of ³Hthymidine incorporation (cpm) versus protein concentration (nM). ³Hthymidine incorporation, which is indicative of the degree of cellproliferation, is measured by standard techniques. These resultsdemonstrate that, in one aspect, mutant CTLA-4-Ig fusion proteins of theinvention have significantly higher potency than Orencia® and/orLEA29Y-Ig proteins in inhibiting or suppressing memory T cellproliferation in vitro (e.g., mutant CTLA-4-Ig proteins have a greaterability than Orencia® and/or LEA29Y-Ig proteins in inhibiting orsuppressing memory T cell proliferation in vitro in human PBMCproliferation assays (recall antigen stimulation)).

This PBMC proliferation assay with PPD antigen stimulation was performedon a number of other mutant CTLA-4-Ig fusion proteins of the invention.Table 8 provides a summary of the data for a set of exemplary mutantCTLA-4-Ig fusion proteins of the invention. Table 8 presents comparisonsof mean IC50 values (nanomolar (nM)) for exemplary mutant CTLA-4-Igfusion proteins versus reference controls (Orencia® and LEA29Y-Ig fusionproteins) using the PBMC proliferation assay with recall antigenstimulation. IC50 values from individual experiments were averaged toprovide mean IC50 values, which were used for statistical analyses. Theterm “SD (mean log IC50)” represents the standard deviation in mean logIC50 values.

TABLE 8 Summary of Data From Exemplary PBMC Proliferation Assays UsingPPD Antigen Stimulation Fusion Protein Mean IC50 Mean Log IC50 SD (MeanLog (dimer) (nM) (nM) IC50) (nM) Orencia ® fusion 5.92 0.67 0.37 proteindimer LEA29Y-Ig 0.31 −0.71 0.53 D3-IgG2 0.03 −1.49 NA D3-12-IgG2 0.06−1.27 0.17 D3-14-IgG2 0.07 −1.17 0.15 D3-17-IgG2 0.07 −1.24 0.25D3-20-IgG2 0.13 −0.91 0.18 D3-27-IgG2 0.17 −0.84 0.30 D3-29-IgG2 0.07−1.16 0.18 D3-34-IgG2 0.51 −0.29 NA D3-50-IgG2 0.28 −0.55 NA D3-53-IgG20.05 −1.29 NA D3-54-IgG2 0.01 −2.00 NA D3-56-IgG2 0.01 −1.92 NAD3-69-IgG2 0.01 −2.00 NA D3-71-IgG2 0.01 −1.87 NA D3-75-IgG2 0.01 −2.02NA D3-76-IgG2 0.01 −1.89 NA Note: The term “NA” in Table 8 means “notavailable”.

Statistical analyses revealed that all mutant CTLA-4-Ig fusion proteinstested were statistically superior in potency to both Orencia® andLEA29Y-Ig fusion proteins with p<0.05, as determined by 1 way ANOVA withpost hoc Dunnett and Bonferroni tests, respectively, discussed supra(e.g., the mutant CTLA-4-Ig fusion proteins have a greater ability thanOrencia® and/or LEA29Y-Ig fusion proteins in inhibiting or suppressingmemory T cell proliferation in vitro in human PBMC proliferation assays(recall antigen stimulation)).

It is believed that given the increased abilities of mutant CTLA-4-Igfusion proteins of the invention to suppress or inhibit proliferation ofmemory T cells (e.g., human memory T cells) in in vitro assays ascompared to Orencia® and/or LEA29Y-Ig fusion proteins, such mutantproteins should also exhibit increased immunosuppressive potencies in invivo therapeutic and/or prophylactic methods or applications as comparedto Orencia®, LEA29Y-Ig, and/or human CTLA-4-Ig (e.g., human CTLA-4-IgG2)fusion proteins. In one aspect, mutant CTLA-4-Ig proteins of theinvention are believed to have a greater ability to suppress or inhibitproliferation of memory T cells (e.g., human memory T cells) in an invivo method or application compared to Orencia®, LEA29Y-Ig, and/or humanCTLA-4-Ig (e.g., human CTLA-4-IgG2) fusion proteins, such as in, e.g.,therapeutic and/or prophylactic methods for suppressing or inhibiting animmune response in a subject (e.g., in the in vivo treatment of immunesystem diseases or disorders in, e.g., a mammal, such as, e.g., ahuman), methods for suppressing or inhibiting rejection of a tissue ororgan transplant from a donor by a recipient (e.g., by a mammal, suchas, e.g., a human), and/or other treatment or diagnostic methodsdescribed elsewhere herein.

Example 8 Measuring Biological Activity of Mutant CTLA-4-Ig MoleculesUsing Human MLR (Mixed Lymphocyte Reaction) Assays

CTLA-4-Ig and variants thereof are potent inhibitors of primaryalloresponses in vitro (Vaughan, A. N. et al., J. Immunol. 165:3175-3181(2000); Wallace P. M., et al., Transplantation 58:602-610 (1994)). Tomeasure improved activity of mutant CTLA-4-Ig proteins in such assays, ahuman mixed lymphocyte reaction (MLR) cell proliferation assay wasdeveloped.

Human blood (freshly collected from a human donor program) was dilutedwith an equal volume of PBS and fractionated to isolate PBMCs using aHistopaque (Sigma, #10771) Ficoll gradient as per the manufacturer'srecommended conditions. PBMC from one donor were diluted in RPMI medium(Sigma, #R8758) supplemented with 10% FBS (Hyclone #SV30014.03) and1×PSG (Invitrogen, #10378-016) and added to 96-well culture plates (BDBiosciences, #353077) at a density of 1×10⁵ cells/well. PBMC from adifferent donor were irradiated at 2500 rads, diluted in the same mediaand added to the same plates at a density of 1×10⁵ cells/well. Testcompounds were serially diluted in the same medium and added to wells inquadruplicate. After incubation at 37° C. for 5 d, ³H thymidine (GEHealthcare, #TRK758-5MCI) was added at 1 μCi/well and plates wereincubated at 37° C. for an additional 18 hr. Cells were harvested with acell harvester (FilterMate Omnifilter-96 Harvester, Perkin Elmer) usingthe manufacturer's recommended conditions and measured for ³H thymidineincorporation using a scintillation counter (Wallac Trilux, #1450-421).Cell proliferation data was analyzed with GraphPad Prism 5 softwareusing a non-linear regression curve fit model (sigmoidal dose-response,variable slope) and the least squares fit method. The IC50 parametersand their associated 95% confidence intervals are reported.

FIG. 9 shows cell proliferation curves from representative MLRproliferation assays involving an exemplary mutant CTLA-4-Ig fusionprotein of the invention: D3-IgG2. Orencia® and LEA29Y-Ig fusionproteins were included as controls for comparison. The graph is a plotof ³H thymidine incorporation (cpm) versus protein concentration (nM).³H thymidine incorporation, which is indicative of the degree of cellproliferation, is measured by standard techniques. These resultsdemonstrate that D3-IgG2 has significantly higher potency than Orencia®and/or LEA29Y-Ig fusion proteins in inhibiting or suppressing primaryallostimulation of T cells in vitro (e.g., D3-IgG2 has a greater abilityto suppress or inhibit primary allostimulation of T cell proliferationin an in vitro MLR assay than Orencia® or LEA29Y-Ig fusion proteins).

This MLR assay was performed on a number of other mutant CTLA-4-Igfusion proteins of the invention. Table 9 provides a summary of the datafor a set of exemplary mutant CTLA-4-Ig fusion proteins of theinvention. Table 9 presents comparisons of mean IC50 values (nanomolar(nM)) for exemplary mutant CTLA-4-Ig fusion proteins versus referencecontrols (Orencia® and LEA29Y-Ig fusion proteins) in the MLR assay. IC50values from two separate experiments were averaged to provide mean IC50values. The term “SD (mean log IC50)” represents the standard deviationin mean log IC50 values. The mean IC50 values for the mutant CTLA-4-Igfusion proteins shown in Table 9 were lower than the respective meanIC50 values of Orencia® and LEA29Y-Ig fusion proteins.

In one aspect, the invention provides mutant CTLA-4-Ig fusion proteinsthat are believed to be superior in potency to Orencia® and/or LEA29Y-Igfusion proteins (e.g., the mutant CTLA-4-Ig fusion proteins are believedto have a greater ability to suppress or inhibit primary allostimulationof T cell proliferation in an in vitro MLR assay than Orencia® and/orLEA29Y-Ig fusion proteins).

It is believed that based on the expected enhanced abilities of mutantCTLA-4-Ig fusion proteins of the invention to suppress or inhibitprimary allostimulation of T cells (e.g., human T cells) in in vitroassays as compared to Orencia® and/or LEA29Y-Ig fusion proteins, suchmutant proteins should also exhibit increased immunosuppressivepotencies in in vivo therapeutic and/prophylactic methods orapplications as compared to Orencia® and/or LEA29Y-Ig fusion proteins.

In one aspect, mutant CTLA-4-Ig fusion proteins of the invention havesignificantly higher potency than Orencia®, LEA29Y-Ig, and/or humanCTLA-4-Ig (e.g., human CTLA-4-IgG2) fusion proteins in inhibiting orsuppressing primary allostimulation of T cells in vitro (e.g., mutantCTLA-4-Ig fusion proteins have a greater ability to suppress or inhibitprimary allostimulation of T cell proliferation in an in vitro MLR assaythan Orencia®, LEA29Y-Ig, and/or human CTLA-4-Ig) fusion proteins. Inone aspect, a mutant CTLA-4-Ig of the invention is believed to have agreater ability to suppress or inhibit primary allostimulation of Tcells (e.g., human T cells) in an in vivo method or application comparedto Orencia®, LEA29Y-Ig, and/or human CTLA-4-Ig fusion proteins, such asin, e.g., a therapeutic and/or prophylactic method of suppressing orinhibiting an immune response in a subject (e.g., in the in vivotreatment of immune system diseases or disorders in a mammal, such ase.g., a human), a method of suppressing or inhibiting rejection of atissue or organ transplant from a donor by a recipient (e.g., by amammal, such as e.g., a human), and/or other treatment or diagnosticmethods described elsewhere herein.

TABLE 9 Summary of Data From Exemplary MLR Assays Fusion Protein MeanIC50 Mean Log IC50 SD (Mean Log (dimer) (nM) (nM) IC50) (nM) Orencia ®fusion 12.53 1.05 0.23 protein dimer LEA29Y-Ig 0.92 −0.19 0.48 D3-IgG20.06 −1.26 NA D3-12-IgG2 0.09 −1.11 0.34 D3-14-IgG2 0.08 −1.20 0.43D3-17-IgG2 0.09 −1.15 0.43 D3-20-IgG2 0.14 −1.11 0.64 D3-27-IgG2 0.13−1.29 0.86 D3-29-IgG2 0.11 −1.14 0.54 D3-34-IgG2 0.10 −1.09 0.39D3-53-IgG2 0.02 −1.64 0.11 D3-54-IgG2 0.07 −1.13 NA D3-56-IgG2 0.06−1.23 NA D3-69-IgG2 0.04 −1.41 NA D3-71-IgG2 0.05 −1.32 NA D3-75-IgG20.08 −1.11 NA D3-76-IgG2 0.08 −1.11 NA Note: The term “NA” in Table 9means “not available”.

Example 9

An adult human patient suffering from rheumatoid arthritis may betreated with a soluble mutant CTLA-4-Ig fusion protein as follows. Apharmaceutical composition comprising a soluble mutant CTLA-4-Ig fusionprotein of the invention and a pharmaceutically acceptable excipient orcarrier (e.g., PBS) is prepared. An exemplary soluble mutant CTLA-4-Igfusion protein comprises two identical monomeric mutant CTLA-4-Ig fusionproteins linked together by one or more disulfide bonds, wherein eachsuch monomeric fusion protein comprises a mutant CTLA-4 ECD polypeptideof the invention comprising a polypeptide sequence selected from any ofSEQ ID NOS:1-73 fused at its C-terminus to the N-terminus of a humanIgG2 Fc polypeptide. Exemplary fusion proteins include those comprisingpolypeptide sequences set forth in any of SEQ ID NOS:74-79, 197-200,205-214, and 219-222. Such fusion proteins are typically expressed indimeric form. The concentration of the fusion protein in thepharmaceutical composition may be in a range of from about 0.05 mg/ml toabout 200 mg/ml, from about 1 mg/ml to about 150 mg/ml, from about 25mg/ml to about 120 mg/ml, from about 1 mg/ml to about 100 mg/ml, fromabout 25 mg/ml to about 100 mg/ml, from about 50 mg/ml to about 100mg/ml, from about 50 mg/ml to about 75 mg/ml, from about 100 mg/ml toabout 150 mg/ml, and the like. For example, the fusion proteinconcentration in the pharmaceutical composition may be about 1 mg/ml, 5mg/ml, 10 mg/ml, 15 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 150 mg/ml, or 200 mg/ml.The pH of such pharmaceutical composition is about pH 4 to about pH 10,including about pH 5 to about pH 9, about pH 6.5 to about pH 8.5,preferably about pH 6.0 to pH 8.0, about 6.5 to pH 7.5, or about pH 7.0to about pH 8.0.

Treatment of the patient's rheumatoid arthritis is carried out byadministering a therapeutically effective amount of the mutant CTLA-4-Igto the patient (e.g., effective dose) by intravenous or subcutaneousinjection. The site of injection may be, e.g., the patient's arm, torso,or leg. The effective dose of the mutant CTLA-4-Ig fusion proteinadministered is typically, but not limited to, e.g., from about 0.01mg/kg to about 100 mg/kg body weight of the adult human patient, suchas, e.g., from about 0.01-5.0 mg/kg, about 0.01-3.0 mg/kg, about0.05-2.5 mg/kg, about 0.1-2.0 mg/kg, about 0.1-1.0 mg/kg, about0.01-0.05 mg/kg, about 0.5-1.5 mg/kg, about 1.0-4.0 mg/kg, about 1.0-3.0mg/kg, about 1.0-2.0 mg/kg, including, but not limited to, about 0.01mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25mg/kg, 0.3 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.25 mg/ml, 1.5 mg/kg,2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 25mg/kg, 50 mg/kg, 75 mg/kg, or 100 mg/kg body weight of the patient isadministered to the patient. Alternatively, an effective amount or doseor dose range described in the “Methods of the Invention” section abovemay be used. The dose of the fusion protein to be administered isdetermined based on the potency of the fusion protein and/or theseverity of the patient's symptoms or signs of rheumatoid arthritis. Thetotal amount of mutant CTLA-4-Ig fusion protein administered to thepatient may be, e.g., from about 0.01 mg to about 100 mg, typically fromabout 1 mg to 100 mg, from about 10 mg to 100 mg, from about 10 mg toabout 75 mg, or from about 10 to about 50 mg. The volume ofpharmaceutical composition administered to the patient is determinedbased upon the concentration of fusion protein in the composition andthe dose of fusion protein to be administered. For subcutaneousinjection, one to two milliliters of the pharmaceutical compositioncomprising the fusion protein is typically administered per injection.For intravenous injection, an appropriate volume of the pharmaceuticalcomposition comprising the fusion protein can be administered.

Following injection of the initial dose, a second identical dose of thefusion protein may be administered to the patient subcutaneously (e.g.,s.c. injection) or intravenously (e.g., i.v. injection) at, e.g., 1, 2,3, or 4 weeks after the initial dose. The dosing schedule may be onedose every two weeks, one dose/month, one dose every two months, etc.depending, e.g., upon the patient's condition. Subsequent doses may beadministered every four weeks or more or less frequently, as necessary.The frequency of dosing may vary depending upon the patient's conditionand may depend upon the severity of the patient's symptoms or signs ofrheumatoid arthritis.

In an exemplary aspect, an amount of a pharmaceutical compositioncomprising a mutant CTLA-4-Ig fusion protein dimer of the invention(such as D3-29-IgG2, D3-54-IgG2, D3-56-IgG2, D3-69-IgG2, etc.) and apharmaceutically acceptable excipient sufficient to provide a dose ofthe fusion protein dimer of about 0.5 mg/kg body weight is administeredby subcutaneous injection to a human suffering from rheumatoid arthritisonce per week or once per month, as needed, depending upon the patient'scondition and response to the drug.

In another exemplary aspect, an amount of a pharmaceutical compositioncomprising a CTLA-4-Ig fusion protein dimer of the invention (such asD3-29-IgG2, D3-54-IgG2, D3-56-IgG2, D3-69-IgG2, etc.) and apharmaceutically acceptable excipient sufficient to provide a dose ofthe fusion protein dimer of about 10 mg/kg is administered intravenouslyto a human suffering from rheumatoid arthritis once per week or once permonth, as needed, depending upon the patient's condition and response tothe drug. Standard i.v. procedures can be used for such administration.For example, the pharmaceutical composition can be infused into thehuman with another fluid, such as a sterile saline solution, dextrosesolution, or other isotonic solution, using a standard continuousintravenous drip through a standard intravenous access device.

Each above-described treatment method by s.c. or i.v. injection isexpected to reduce or alleviate one or more signs, symptoms, orbiological responses associated with rheumatoid arthritis, such as,e.g., inflammation, joint tenderness, joint swelling, pain, tissueatrophy, and stiffness, in the patient. Such treatment may reducefurther progression of the disease in the patient, particularly in theconnective, muscular, and skeletal tissues. For example, such treatmentmay reduce the progression of damage to or deterioration of connectivetissue, atrophy muscular tissue, bone, joints, cartilage, and/or spinalcolumn, and the like in the patient. Additional clinical symptoms of thedisease, including damage caused to the skin, central nervous system ororgans, may also be lessened or alleviated. Such treatment may alsoimprove physical functioning of the patient.

Example 10

An adult human patient undergoing maintenance therapy for prevention oforgan transplant rejection may be treated with a soluble mutantCTLA-4-Ig fusion protein as follows. A pharmaceutical compositioncomprising a soluble mutant CTLA-4-Ig fusion protein of the inventionand a pharmaceutically acceptable excipient or carrier (e.g., PBS or thelike) is prepared. An exemplary soluble mutant CTLA-4-Ig fusion proteincomprises two identical monomeric mutant CTLA-4-Ig fusion proteinslinked together by one or more disulfide bonds, wherein each suchmonomeric fusion protein comprises a mutant CTLA-4 ECD polypeptide ofthe invention comprising a polypeptide sequence selected from any of SEQID NOS:1-73 fused at its C-terminus to the N-terminus of a human IgG2 Fcpolypeptide. Exemplary fusion proteins include those comprisingpolypeptide sequences set forth in any of SEQ ID NOS:74-79, 197-200,205-214, and 219-222. The concentration of the fusion protein in thepharmaceutical composition may be in a range of from about 0.05 mg/ml toabout 200 mg/ml, from about 1 mg/ml to about 150 mg/ml, from about 25mg/ml to about 120 mg/ml, from about 1 mg/ml to about 100 mg/ml, fromabout 25 mg/ml to about 100 mg/ml, from about 50 mg/ml to about 100mg/ml, from about 50 mg/ml to about 75 mg/ml, from about 100 mg/ml toabout 150 mg/ml, and the like. For example, the fusion proteinconcentration in the pharmaceutical composition may be about 1 mg/ml, 5mg/ml, 10 mg/ml, 15 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 150 mg/ml, or 200 mg/ml.The pH of such pharmaceutical composition is about pH 4 to about pH 10,including about pH 5 to about pH 9, about pH 6.5 to about pH 8.5, aboutpH 6.0 to pH 8.0, about 6.5 to pH 7.5, or about pH 7.0 to about pH 8.0.

Maintenance therapy for prevention or suppression of organ transplantrejection is carried out by administering a therapeutically effectiveamount of the mutant CTLA-4-Ig to the patient (e.g., effective dose) whohas received an organ transplant (e.g., kidney transplant) byintravenous or subcutaneous injection. The site of injection may be,e.g., the patient's arm, torso, or leg. The effective dose of the mutantCTLA-4-Ig fusion protein administered is typically, but not limited to,e.g., from about 0.01 mg/kg to about 100 mg/kg body weight of the adulthuman patient, such as, e.g., from about 0.01-5.0 mg/kg, about 0.01-3.0mg/kg, about 0.05-2.5 mg/kg, about 0.1-2.0 mg/kg, about 0.1-1.0 mg/kg,about 0.01-0.05 mg/kg, about 0.5-1.5 mg/kg, about 1.0-4.0 mg/kg, about1.0-3.0 mg/kg, about 1.0-2.0 mg/kg, including, but not limited to, about0.01 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg,0.25 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.25 mg/ml, 1.5mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 20mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, or 100 mg/kg body weight of thepatient is administered to the patient. Alternatively, an effectiveamount or dose or dose range described in the “Methods of the Invention”section above may be used. The dose of the fusion protein to beadministered is determined based on the potency of the fusion proteinand/or the severity of the patient's symptoms or signs of organtransplant rejection. The total amount of mutant CTLA-4-Ig fusionprotein administered to the patient may be, e.g., from about 0.01 mg toabout 100 mg, typically from about 1 mg to 100 mg, from about 10 mg to100 mg, from about 10 mg to about 75 mg, or from about 10 to about 50mg. The volume of pharmaceutical composition administered to the patientis determined based upon the concentration of fusion protein in thecomposition and the dose of fusion protein to be administered. Thefusion protein can be administered on any day post-transplant, e.g., day1, 4, 7, 14, 28, 56, 84, etc. post-transplant. For subcutaneousinjection, one to two milliliters of the pharmaceutical composition istypically administered. For intravenous injection, an appropriate volumeof the pharmaceutical composition comprising the fusion protein can beadministered.

Following injection of the initial dose, a second identical dose of thefusion protein may be administered to the patient subcutaneously orintravenously at 1, 2, 3, or 4 weeks after the initial dose. The dosingschedule may be one dose every two weeks, one dose/month, one dose everytwo months, etc. depending, e.g., upon the patient's condition.Subsequent doses may be administered every four weeks or more or lessfrequently, as necessary, and continued, if desired, on a monthly basis.The frequency of dosing may vary depending upon the patient's conditionand may depend upon the severity of the patient's symptoms or signs oforgan transplant rejection.

Such treatment is expected to reduce or alleviate one or more signs,symptoms, or biological responses associated with organ transplantrejection, such as, e.g., acute rejection of the transplanted organ,chronic rejection of the transplanted organ, decrease in function of thetransplanted organ, increase in serum creatinine levels in the patient,and/or increased infiltration of T cells into the transplanted organ.Such treatment may reduce the likelihood of rejection of thetransplanted organ by the patient's immune system.

Example 11 Pharmacokinetic Assessment of Mutant CTLA-4-IgG2 FusionProteins in Rats

The serum concentration of a therapeutic after external administrationto a living organism greatly influences therapeutic efficacy, and isdetermined by pharmacokinetic (PK) evaluation. In the followingprocedure, PK profiles were assessed in rats for representative mutantCTLA-4-IgG2 test articles D3-29-IgG2, D3-54-IgG2, D3-56-IgG2,D3-69-IgG2, and D3-75-IgG2 in comparison to both hCTLA-4-IgG2 andOrencia® fusion protein test articles. The study design, and datageneration and interpretation are described forthwith.

In Life Study Design.

Weight-matched, male Hans Wistar rats were used after an acclimatizationperiod of at least 5 days. Test article dosing volumes were calculatedfor individual animals based on their weight such that all received 1mg/kg of test article. A typical 150 gram (g) rat therefore received 150μl dosing volume since every test article was prepared at 1 mg/ml inPBS. A single administration of a mutant CTLA-4-IgG2 test articledescribed above, hCTLA-4IgG2, or Orencia® fusion protein was delivered,either as an intravenous (i.v. or IV) bolus, or by the subcutaneous(abbreviated as “s.c.” or “SC”) route. The study size was sufficient fora minimum of four blood samplings of 300 μl per timepoint, whilerestricting the blood volume removed from any given rat to no more than10% total blood volume. The blood sampling time course was eitherpre-dose, 5 minutes (min), 30 min, 2 hours (h), 4 h, 8 h, 1 day (d), 2d, 3 d, 4 d, 6 d, 8 d, 10 d, 12 d, and 14 d post-dose, or 5 min, 30 min,2 h, 4 h, 8 h, 1 d, 2 d, 3d, 4 d, 6 d, 8d, 10 d, 11 d, 12 d, 13 d, 14 dand 15 d post-dose for i.v and s.c. administrations, respectively. Serumwas prepared from individual blood samples, and tested by ELISA toquantify the presence of the administered test article.

PK ELISA Method.

The mutant CTLA-4-IgG2, hCTLA-4IgG2, or Orencia® fusion protein presentin the serum samples were bound to human CD80-murine IgG (describedabove) pre-coated onto microtiter plates. Detection was achieved byaddition of a Horse Radish Peroxidase (HRP) conjugated goat anti-humanIgG (Jackson ImmunoResearch #109-035-098). Quantification was throughthe use of a chromogenic HRP substrate, 3,3′,5,5′-tetramethylbenzidine(TMB), plus hydrogen peroxide (Kem-En-Tec #4390A), whereby the reactionwas stopped by addition of 0.2N sulphuric acid (H₂SO₄), and opticalabsorbance was measured at 450 nm by a spectrophotometer. Serum sampleswere pre-diluted 1/20 before addition to the ELISA plate such thatmatrix was normalized to 5% rat sera, and further diluted in 5% rat serafor a total of eight dilutions. The concentration of each diluted serumsample was quantified against a standard curve prepared using titrationsof the same test article spiked into 5% rat sera. The standard curveranged from 10 to 0.078 ng/ml, and the accuracy range was determined tobe from twice background to 5 ng/ml in 5% rat sera. The quality of thestandard curve was assessed by quality controls (QC) of the same testarticle prepared in 5% rat sera at high, middle and low concentrationsof the standard curve accuracy range. The QC acceptance criterion wasfor the observed QC concentration to be within 20% of the expected QCconcentration. The concentration of an individual unknown serum samplewas generated by averaging the concentrations assigned to dilutions withoptical densities (ODs) within the accuracy range of the standard curve.At least 4 individual serum samples were used to calculate the meanserum concentration at each nominal timepoint.

PK Parameters.

FIGS. 15A and 15B show PK profiles for Orencia® fusion protein,hCTLA-4-IgG2, and the mutant CTLA-4-IgG2 fusion proteins administered at1 mg/kg as a single (A) intravenous (IV) bolus or (B) subcutaneous (SC)injection in rats. The error bars represent standard deviation of themean (SD). The dashed line represents the lower limit of quantificationfor the ELISA (˜3 ng/ml for test article in 100% sera). The mean serumconcentration at each nominal timepoint after test articleadministration at time 0 h comprises the datapoints of the semi-logconcentration-time profile in FIG. 15.

The mean serum concentrations were used to generate PK parameters usingWinNonLin model 201 (i.v.—bolus input), or model 200 (extravascularinput) for the i.v. (IV) or s.c. (SC) dosing routes, respectively. Table10 summarizes the key PK parameters for s.c. and i.v. administrationroutes, respectively.

TABLE 10 Summary of PK Parameters for Orencia ® fusion protein,hCTLA-4-IgG2, and Mutant CTLA-4-IgG2 Fusion Proteins Administered at 1mg/kg as a Single I.V. or S.C. Bolus to Rats Cmax T½ AUC Cl Vz CompoundRoute (ug/L) (h) (h*ng/L) (ml/h/kg) (ml/kg) Orencia SC 3.3 70.0 391 2.6258.13 CTLA-4IgG2 SC 5.9 45.0 838 1.2 77.49 D3-29 SC 4.6 11.2 650 1.524.90 D3-54 SC 4.5 23.6 528 1.9 64.52 D3-56 SC 7.4 23.7 1049 1.0 32.67D3-69 SC 6.0 28.1 967 1.0 41.97 D3-75 SC 9.0 56.4 1204 0.8 67.59 OrenciaIV 22.3 42.3 728 1.37 83.9 CTLA-4IgG2 IV 66.2 49.8 2483 0.40 29.0 D3-29IV 43.0 33.0 912 1.10 52.2 D3-54 IV 20.6 39.4 1034 0.97 55.0 D3-56 IV34.2 15.3 1822 0.55 12.1 D3-69 IV 81.9 83.0 2133 0.47 56.1 D3-75 IV 30.165.4 2326 0.43 40.5

Cmax refers to the maximum serum concentration of test article. Theterminal half-life value (T½) is the time taken in hours for theconcentration of test article in the serum to decline by half during thetermination phase of the concentration-time profile. The area under theserum concentration-time curve (AUC) is quantified from time zero toinfinity using the trapezoidal rule. The clearance (Cl) was calculatedusing the equation dose/AUC, whilst the volume of distribution (Vz) ofthe terminal phase was calculated using the equation Cl/k.

The bioavailability factor (F) for each compound is presented in Table11, and was determined by calculating the AUC by the subcutaneousroute/AUC by the intravenous route.

TABLE 11 Bioavailability Comparison of Orencia ® Fusion Protein,hCTLA-4-Ig2, D3-29-IgG2, D3-54-IgG2, D3-56-IgG2, D3-69-IgG2, andD3-75-IgG2 Fusion Proteins Compound Bioavailability Orencia 0.54CTLA-4IgG2 0.34 D3-29 0.71 D3-54 0.51 D3-56 0.58 D3-69 0.45 D3-75 0.52

Human CTLA-4IgG2 and Orencia® fusion protein displayed similar PKprofiles despite their difference in IgG frameworks, implying that thehuman IgG2 and mutated human IgG1 Fc portions of these respective testarticles have comparable PK activity when the functional domain isconstant. By inference, any changes seen in PK profile for mutantCTLA-4-IgG2 fusion proteins as compared to Orencia® fusion proteinshould therefore be attributable to differences in the functionaldomain, and not the Fc portion.

For each mutant CTLA-4-IgG2 fusion protein, elimination was slow assuggested by the long half-life values and large area under the curves.Half-lives ranged from 11.2 to 56.4 hr for SC dosing, and from 15.3 to83.0 hours (h) for IV dosing in comparison to Orencia® fusion protein,which had a half-life of 70.0 or 42.3 hours when administered by SC orIV routes, respectively. AUC values for the mutant CTLA-4-IgG2 fusionproteins were on average superior to the Orencia® fusion protein. AfterSC dosing the mean was 879.5+/−281.6 h*kg*ng/L/mg as compared to 391h*kg*ng/L/mg for SC Orencia® fusion protein, while an IV administrationgave an average AUC of 1645.5+/−641.1 h*kg*ng/L/mg as compared to 728h*kg*ng/L/mg for IV Orencia® fusion protein.

The volume of distribution was similar for both routes of administrationin that mutant CTLA-4-IgG2 fusion proteins were distributed outside theserum but inside the extravascular fluid as suggested by the averagevalue of 44.8 ml/kg, which is above the plasma reference volume of 30ml/kg, and within the extracellular fluid reference limit of 300 ml/kgfor a standard rat (Davies, B. et al., Pharm. Res. 10(7):1093-95(1993)).

Bioavailability was also similar for the majority of the mutantCTLA-4-IgG2 fusion proteins, with an average of 0.6+/−0.1, whichcompared favorably to the 0.53 bioavailability of Orencia® fusionprotein.

Finally, Cmax was generally higher for the mutant CTLA-4-IgG2 fusionproteins as compared to the Orencia® fusion protein. Cmax values rangedfrom 4.5 to 9.3 ug/L for dosing, and from 20.6 to 81.9 ug/L for IVdosing in comparison to the Orencia® fusion protein, which had a Cmax of3.3 or 22.3 ug/L when administered by SC or IV routes, respectively.

Overall, the PK data showed that all assessed mutant CTLA-4-IgG2 fusionproteins typically had a non-inferior PK profile to the Orencia® fusionprotein when administered to rats at 1 mg/kg via SC or IV routes, andthat IgG1 or IgG2 Fc portions were comparable when the functional domainwas constant.

Example 12

This example describes a method for creating a stably transfected cellline for expressing mutant CTLA4-Ig fusion proteins of the invention,using the cell line for routine laboratory scale production of themutant CTLA4-Ig fusion protein, and purifying the mutant CTLA-4-Igfusion proteins from cell expression media. Although this examplespecifically describes a method for creating a stably transfected CHO-K1cell line for expressing the D3-54-IgG2 fusion protein, using such cellfor laboratory scale production and purifying the protein from media,the methods described herein can be used with any mutant CTLA-4-Igfusion protein of the invention and/or any appropriate cell linedisclosed above.

Creation of Stably Transfected Cell Line.

Materials.

CHO-K1 naïve (untransfected) cells: CHO-K1 cells adapted to serum-freesuspension growth in chemically-defined medium (Cell ID:M4-PeM-0436-112-01) were stored in liquid nitrogen vapor phase (DewarMVE1536P). One vial of M4-PeM-0436-112-01 was thawed and cultured withCD OptiCHO™ medium (Invitrogen, #12681) in shake flasks. The culturesprovided cells for transfection and conditioned medium for growth andcloning. All cultures were grown at 37° C., 5% CO₂.

Plasmid: DNA encoding the D3-54 mutant CTLA-4 ECD fused to a human IgG2aFc region was inserted into the CET1019AS UCOE vector (Millipore) andthe resulting plasmid CET1019AS-D3-54-IgG2 was used for alltransfections.

Cell culture medium: CD Opti-CHO (Invitrogen #12681) chemically-definedanimal component-free medium, supplemented with 2% v/v 200 mML-glutamine (Invitrogen #25031), was used for all cultures.

Conditioned medium: Conditioned medium was obtained by cultivating theparental CHO-K1 cell line in CD Opti-CHO medium. At cell counts≧5×10⁵cells/ml, the cell culture was centrifuged and the conditioned mediumsupernatant was sterile-filtered. Conditioned medium was made fresh eachday for use or stored at 2-8° C. for up to 7 days.

50% conditioned medium: Conditioned medium (above) combined with anequal volume of fresh cell culture medium; made fresh each day it isused.

Analytical Methods.

Cell count and viability determination were performed with a Cedex orCedex HiRes cell counter (Innovatis).

Initial screening of clones expressing the mutant CTLA-4-Ig fusionprotein (D3-54-IgG2) for production was performed by ELISA. ELISA plateswere coated with hCD80-murine Ig fusion protein overnight. The followingday, samples to be analyzed were transferred to the ELISA plates at 50-and 200-fold dilutions in duplicate. After two hours incubation,anti-human IgG-HRP antibody was added and incubated for 30 minutes. Theplates were developed with TMB and read at 450 nm. Raw optical densities(OD) were reported.

Quantitative determination of the D3-54-IgG2 fusion proteinconcentration was performed by a Protein A HPLC method using a PorosA/20 column (ABI #1-5024-12). Two buffers were used: Buffer A: 50 mMphosphoric acid, 150 mM potassium chloride, pH 7.6±0.1 and Buffer B: 50mM phosphoric acid, 150 mM potassium chloride, pH 2.5±0.1. Both buffersadditionally had 5% isopropanol added. Equilibration and wash aftersample injection was with 42% Buffer A and 58% Buffer B (pH 6.5).Elution was a linear gradient to 12% Buffer A and 88% Buffer B over 1minute.

Procedure.

The naïve adherent CHO-K1 cells used for the creation of stable celllines used in GMP production of D3-54-IgG2 fusion protein were adaptedto suspension growth in chemically defined CD OptiCHO™ medium. A vial ofthese naïve CHO-K1 cells was thawed and grown in 125 ml shake flaskscontaining CD OptiCHO™ medium to a density of 5×10⁵ viable cells/ml.2×10⁶ viable cells were resuspended in 400 μl of 50% conditioned mediumand combined with 20 μg plasmid DNA (D3-54-IgG2 in a CET1019AS UCEOvector) in a cuvette. Electroporation was performed with a Gene XcellPulser (BioRad) at 320 volts (V) with a square wave pulse length of 15milliseconds (ms). Duplicate transfections were performed and then thecells were pooled. The pooled cells were transferred to a T-25 flaskcontaining 5 ml of 50% conditioned medium and incubated for two days.

Transfected cells were either directly dispensed into 96-well plates forcloning or cultured with antibiotic selection until a stable pool wasobtained, and then dispensed into 96-well plates. Two days afterelectroporation, the culture was diluted to 1250 cells/ml in conditionedmedium containing 8 μg/ml of puromycin for selection pressure. The cellswere dispensed into 96-well plates at 200 μl per well (250 cells/well).The plates were incubated for approximately 10 days to killuntransfected and transiently-transfected cells. After 10-12 days, everywell in every plate was visually inspected to identify wells with singlecolonies. These wells were later re-inspected to verify they containedsingle, healthy colonies suitable for expansion into 24-well plates.

Two days after electroporation, the culture was centrifuged andresuspended in 50% conditioned medium containing 7 μg/ml of puromycinfor selection pressure. A control flask was also inoculated withuntransfected cells in the same medium. Based on on-going optimizationstudies, the puromycin concentration was increased to 8 μg/ml after 3days. The stable pool was generated 10-12 days after selection, when allcells in the control flask died. Product expression in the stable poolwas verified by protein A HPLC and culture viability was verified tobe >95%. The cells were serially diluted in conditioned medium withoutpuromycin to a final density of 3.8 cells/ml. The cells were seeded onto96-well plates at 200 μl per well (75 cells/plate or 0.8 cells/well).

After one day every well in every plate was visually inspected toidentify wells with single cells or colonies. The next day, wells withsingle colonies of 2-4 cells were selected. Any wells with more than twocolonies were eliminated. A second operator verified the selections. Thewells were later re-inspected to verify they contained single, healthycolonies suitable for expansion into 24-well plates

Clones from 96-well plates were expanded into 24-well plates containing1 ml of conditioned medium per well with 8 μg/ml of puromycin. Theentire contents of the selected wells from the 96-well plates withsingle colonies were transferred to individual wells in the 24-wellplates. 200 μl was taken from each new well in the 24-well plate to washthe corresponding well in the 96-well plate and transferred back. Forbackup, 200 μl of conditioned medium containing 8 μg/ml of puromycin wasadded back to each well in the 96-well plates.

After 1-3 days, each well in the 24-well plates was sampled and testedfor D3-54-IgG2 expression by ELISA. Clones for further expansion wereselected based on the raw ELISA OD values as well as demonstration ofadequate growth.

The top 35-40 clones, based on ELISA and observable growth results wereexpanded into T-25 flasks containing 5 ml of conditioned medium with 8μg/ml of puromycin. The entire contents of each of the selected wellsfrom the 24-well plates were transferred to individual T-25 flasks.Residual cells in the wells were washed with the same medium and addedto the corresponding T-25 flask. For backup, 1 ml of conditioned mediumcontaining 8 μg/ml of puromycin was added back to each well in the24-well plates.

The number of clones was further reduced by selecting for clones withthe highest productivity. Cells were resuspended in fresh medium at1-2×10⁵ viable cells/ml and seeded into T25 flasks (5 ml culture) or 125ml shake flasks (12 ml culture). The cultures were incubated for 22-24hours and then a final cell density and viability determination was madeand a sample was taken for product concentration determination byProtein A HPLC. Productivity was calculated by dividing the total amountof protein produced by the total number of viable cells in the flask anddividing by the culture duration. The units were converted to picogramsper cell per day or “pcd”. Clones were ranked according to their pcdvalues, but clones that did not exhibit significant growth were omitted.The top clones were expanded to 125 ml shake flasks for cryopreservationand further evaluation of growth and productivity.

Clones to be further evaluated were seeded into 250 ml shake flasks at1×10⁵ viable cells/ml in 50 ml fresh medium. When the cell densityreached 1×10⁶ viable cells/ml, the culture was passaged into a new shakeflask, again at 1×10⁵ viable cells/ml in 50 ml fresh medium. Thepassaging was repeated once more. During this third passage, the culturewas sampled daily for cell count, viability, and product concentrationdetermination by Protein A HPLC.

The top clones expressing D3-54-IgG2 fusion protein based on the growthand specific production rates were selected for subcloning. Subcloningwas performed by limiting dilution as described in the section above forstable pools, with the exception that puromycin was not used at anytime. Expansion, screening, and evaluation of subclones were alsoperformed as described above.

Selected subclones were repeatedly passaged in shake flasks forapproximately 90 days to evaluate production stability. At each passage,cells were seeded in 125 ml shake flasks at 1×10⁵ viable cells/ml in 25ml fresh medium. The cultures were passaged every 3-4 days. Before eachpassage, the cell density and viability were measured and a sample wastaken for product concentration determination by Protein A HPLC.

Selected clones and subclones were cryopreserved from the time ofranking by pcd values to various points in the stability evaluation.Freezing medium was freshly made with 90% growth medium and 10% DMSO(Sigma). Cells from shake flask cultures were centrifuged andresuspended in freezing medium at densities ranging from 2-10×10⁶cells/ml. The cell suspension was dispensed in 1 ml aliquots intocryogenic vials. The cryogenic vials were placed in an isopropanolfreezing containers and stored at −80° C. overnight. The frozen vialswere transferred to liquid nitrogen vapor phase storage the followingday.

Production of Mutant CTLA4-IgG2 Fusion Proteins.

A cryovial containing 1 ml volume of CHO-K1 cells expressing D3-54-IgG2fusion protein is thawed and grown in 125 ml shake flasks containing CDOptiCHO™ medium at 37° C. and 5% CO₂ to a density of 5×10⁵ viablecells/ml. Several flasks are combined to inoculate a wave bag at 1-2×10⁵cells/ml in a 5 or 10 L volume of CD OptiCHO™ medium with 4 mMglutamine. The wave bag culture is maintained in a 37° C. incubatorsupplemented with 5% CO₂ at a rocking platform setting of 18-22 rpm andangle of 8 degrees for equipment purchased from Sartorius StedimBiotech. The culture is sampled daily for cell count, viability,nutrient level, metabolite profile and expression level of the mutantCTLA-4-IgG2 (i.e., D3-54-IgG2) using a Protein A HPLC assay. The cultureis harvested when the viability declines to ˜50% typically 9-11 dayspost inoculation. The cell culture material is clarified by filtrationusing a combination of depth filtration and sterile filtration andeither used immediately for further processing or stored at 2-8° C.

Purification of Mutant CTLA-4-IgG2 Fusion Proteins.

Mutant CTLA4-IgG2 fusion proteins (D3-54-IgG2) were purified byProtein-A affinity chromatography using an AKTA Explorer HPLC system (GEHealthcare). A mutant CTLA-4-Ig fusion protein (D3-54-IgG2) was bound toMabSelect Protein A FF columns (GE Healthcare, #17-5079-01) in PBSbuffer (Invitrogen), loaded at ˜10 mg/ml of chromatography media, washedwith the same buffer, eluted with 100 mM citric acid buffer (pH 4.0),and then neutralized by addition of 1/10 volume of 2M Tris base. TheProtein A purified sample is further processed by diafiltration using atangential flow filtration (TFF) system with a buffer exchange into 20mM Tris-Cl, pH 7.5.

The buffer exchanged protein sample is further purified by anionexchange chromatography on a Q-Sepharose column loaded at 10 mg/mlloading density. The bound protein is eluted using a 20 column volume(CV) linear NaCl gradient from 0-500 mM NaCl in 20 mM Tris-Cl, pH 7.5.Major peak fractions are pooled and the concentration is determined bymeasuring absorbance at 280 nm.

The protein purity is confirmed by SDS-PAGE analysis and the monomercontent is determined using a size exclusion-HPLC method. Samples arestored in aliquots at 2-8° C. or at −20° C. for extended periods priorto use.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. It is understood that the materials, examples, andembodiments described herein are for illustrative purposes only and notintended to be limiting and that various modifications or changes inlight thereof will be suggested to persons skilled in the art and are tobe included within the spirit and purview of this application and scopeof the appended claims. All publications, patent applications, patents,or other documents mentioned herein are incorporated by reference intheir entirety for all purposes to the same extent as if each suchindividual publication, patent, patent application or other documentwere individually specifically indicated to be incorporated by referenceherein in its entirety for all purposes and were set forth in itsentirety herein. In case of conflict, the present specification,including definitions, will control.

1-287. (canceled)
 288. A method of inhibiting or preventing rejection ofa tissue, cell, graft, or organ transplant from a donor by a recipientsubject, the method comprising administering to the recipient subject inneed thereof a therapeutically effective amount of an isolated orrecombinant fusion protein, thereby inhibiting or preventing rejectionof the tissue, cell, graft, or organ transplant by the recipientsubject, wherein the isolated or recombinant fusion protein comprises(a) a first polypeptide comprising a polypeptide sequence that has atleast 96% identity to the polypeptide sequence of SEQ ID NO:50, and (b)a second polypeptide, wherein the second polypeptide comprises a humanimmunoglobulin (Ig) polypeptide, wherein the fusion protein has anability to bind CD80 or CD86 or an extracellular domain of either CD80or CD86, and/or an ability to inhibit or suppress a T cell immuneresponse.
 289. The method of claim 288, wherein the fusion protein, isadministered to the recipient subject prior to, simultaneously with, orafter tissue, cell, graft, or organ transplantation.
 290. The method ofclaim 288, wherein the donor and the recipient subject are human.291-292. (canceled)
 293. The method of claim 288, wherein the effectiveamount comprises from about 0.001 mg/kg weight of the subject to about200 mg/kg weight of the subject. 294-296. (canceled)
 297. The method ofclaim 288, wherein the organ transplant is a kidney transplant, a livertransplant, a lung transplant, or a heart transplant. 298-364.(canceled)
 365. A method of inhibiting or preventing rejection of atissue, cell, graft, or organ transplant from a donor by a recipientsubject, the method comprising administering to the recipient subject inneed thereof a therapeutically effective amount of an isolated orrecombinant fusion protein dimer thereby inhibiting or preventingrejection of the tissue, cell, graft, or organ transplant by therecipient subject, wherein the isolated or recombinant fusion proteindimer comprises two monomeric fusion proteins linked via at least onedisulfide bond formed between two cysteine residues present in eachmonomeric fusion protein, wherein each monomeric fusion proteincomprises (a) a first polypeptide comprising a polypeptide sequencehaving at least 96% identity to SEQ ID NO:50; and (b) a secondpolypeptide comprising human immunoglobulin (Ig) polypeptide, whereinthe fusion protein dimer has an ability to bind CD80 and/or CD86, and/orCD80-Ig and/or CD86-Ig, and/or has an ability to inhibit or suppress animmune response.
 366. The method of claim 365, wherein the fusionprotein dimer is administered to the recipient subject prior to,simultaneously with, or after tissue, cell graft, or organtransplantation.
 367. The method of claim 365, wherein the donor and therecipient subject are human.
 368. The method of claim 365, wherein theeffective amount comprises from about 0.001 mg/kg weight of the subjectto about 200 mg/kg weight of the subject.
 369. The method of claim 365,wherein the organ transplant is a kidney transplant, a liver transplant,a lung transplant, or a heart transplant.
 370. The method of claim 288or 365, wherein the first polypeptide comprises SEQ ID NO:50.
 371. Themethod of claim 370, wherein the fusion protein comprises the sequenceSEQ ID NO:213.
 372. The method of claim 371, wherein the fusion proteinconsists of a sequence selected from the group consisting of SEQ IDNO:213 and SEQ ID NO:199.